TWI675838B - Pyrazolopyrimidinone or pyrrolotriazinone derivatives, preparation method and medical use thereof - Google Patents

Abstract

The invention relates to pyrazolopyrimidones or pyrrolotriazinone derivatives, a preparation method thereof and its application in medicine. Specifically, the present invention relates to a pyrazolopyrimidone or pyrrolotriazinone derivative represented by the general formula (I) and a pharmaceutically acceptable salt thereof, a preparation method thereof, and their use as a gonadotropin-releasing hormone The (GnRH) receptor antagonist is particularly used as a therapeutic agent such as endometriosis, wherein the definition of each substituent in the general formula (I) is the same as that in the specification.

Description

Pyrazolopyrimidones or pyrrolotriazinone derivatives, preparation method and application thereof in medicine

The invention relates to a novel pyrazolopyrimidone or pyrrolotriazinone derivative and a pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the derivative, and a gonadotropin releasing hormone ( GnRH) receptor antagonists are particularly useful as therapeutic agents such as endometriosis.

Endometriosis is a common estrogen-dependent gynecological disease that often occurs during women's reproductive age, and its mechanism of action is unclear. Difficulty in diagnosing endometriosis and complex symptoms such as unknown etiology have seriously prevented the discovery of effective treatment methods. At present, endometriosis is mainly diagnosed by laparoscopy and treated by surgery, or by taking contraceptives, GnRH receptor agonists or progestogens to reduce estrogen levels in the body.

The current incidence of endometriosis is very high. According to Datamonitor 2009 data, only India and China The number of female patients with endometriosis has exceeded 68 million (31.288 million in India, 37.535 million in China), and the number of patients in the seven major market countries has also exceeded 17 million. Datamonitor estimates that from 2009 to 2018, the endometriosis market will grow from US $ 764 million in 2009 (US $ 596 million, EU US $ 117 million, and Japan US $ 51 million) to US $ 1.156 billion in 2018 (US $ 8.44) (US $ 206 million, EU: US $ 206 million, Japan: US $ 106 million), and the Chinese market will have more room for growth.

Gonadoliberin; gonadotropin releasing hormone (GnRH), also known as luteinizing hormone releasing hormone (LHRH), is a decapeptide hormone (pGlu-His-Trp-Ser-Tyr-Gly) synthesized by neuroendocrine cells of the hypothalamus. -Leu-Arg-Pro-Gly-NH ₂ ), is a central regulator in the endocrine reproductive system. It is delivered to the pituitary by the hypothalamic pituitary portal circulatory system, which binds to GnRH receptor cells in the anterior pituitary gland, and gonadotropins such as Luteinizing Hormone (LH) and Follicle-Stimulating Hormone, FSH) secretes and releases, regulates the normal development of the ovary and corpus luteum, and plays an important role in the hypothalamus-pituitary-gonadal axis. GnRH receptors exert their regulatory effects by coupling with G proteins that can activate the second messenger system of phospholipids and inositol calcium, while LH regulates the production of sex steroids, and FSH regulates male spermatogenesis and female follicular development.

LH and FSH are released into the circulatory system and combine with receptors on specific cells of the ovary or testes to stimulate steroid production. In the presence of sex steroids, diseases such as endometriosis, uterine fibroids, and prostate cancer are exacerbated, and long-acting peptide GnRH receptors need to be given Agonists and antagonists are used for therapeutic control.

Peptide GnRH receptor antagonists include GnRH-derived linear peptides (US 5,171,835), cyclic hexapeptide derivatives (US 2002/0065309), bicyclic peptide derivatives (Journal of Medicinal Chemistry, 1993; 36: 3265-73), etc. And peptide GnRH receptor agonists include Leuprorelin (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt). However, peptide compounds have many problems to be solved, including oral absorption, dosage form, dose volume, drug stability, sustained effect, and metabolic stability. The main reason why small molecule GnRH receptor antagonists are superior to existing peptide-based therapies is that small molecule GnRH receptor antagonists can be directly administered orally, which is convenient and fast. Studies have shown that small molecule antagonists have significant effects on hormone-dependent diseases such as endometriosis, precocious puberty, and prostate cancer.

GnRH receptor agonist-mediated indirect tumor suppression mechanism is a long-term action on the hypothalamus-pituitary-gonadal axis, leading to a decrease in pituitary gonadotropin (FSH, LH), thereby reducing the secretion of sex hormones and indirectly inhibiting tumor cells Grow. The GnRH receptor antagonist directly inhibits the release of pituitary gonadotropin, thereby inhibiting the growth of tumor cells.

In view of the limitations of peptide GnRH receptor antagonists, some non-peptide GnRH receptor antagonists have been proposed and entered the development, clinical trial and marketing stages. For example, Elagolix (also known as NBI-56418 or ABT-620) was developed by Abbott and Neurocrine Biosciences Inc, a small-molecule GnRH receptor antagonist, is currently in Phase III clinical trials and is mainly used to treat endometriosis (stage III) and uterine fibroids (II period). In June 2012, the 94th annual meeting of the Endocrine Association of Houston published a clinical phase II data on endometriosis: in 131 patients who received elagolix (150 or 250 mg qd), leuprorelin depot (3.75 mg sc, once a month for 12 weeks) or placebo-treated female endometriosis patients, the serum estrogen in patients treated with elagolix was significantly lower than those treated with leuprorelin and placebo. At the same time, the safety and tolerability of elagolix has been well verified.

Relugolix, also known as TAK-385, is an oral antagonist of the small molecule GnRH receptor developed by Japan's Takada Pharmaceutical Company for the treatment of endometriosis, uterine fibroids and prostate cancer. In 2011, it entered the phase II clinical research phase of endometriosis and uterine fibroids, and launched the phase I clinical research of prostate cancer in the same year.

A series of small molecule GnRH receptor antagonist patents currently published include WO2006096785, WO2010026993, WO2011076687, WO2012175514 and the like.

Although a lot of meaningful research has been carried out in this field, there is still a need to continue research and development of more effective small molecule GnRH receptor antagonists. The present invention provides a novel structure of GnRH receptor antagonists and discovers compounds with such structures It has good activity and can effectively treat endocrine and reproductive system diseases.

其中：當G為N時，D為C，E為-CH-；當G為C時，D和E為N；R¹選自烷基、環烷基、雜環基、芳基、雜芳基或-OR⁶，其中該烷基、環烷基、雜環基、芳基或雜芳基各自獨立地視需要進一步被一個或多個選自鹵素、氰基、硝基、烷基、鹵烷基、羥烷基、烯基、炔基、環烷基、雜環基、芳基、雜芳基、-OR⁶、-C(O)OR⁶、-OC(O)R⁶、-NHS(O)_mR⁶、-C(O)R⁶、-NHC(O)R⁶、-NR⁷R⁸、-OC(O)NR⁷R⁸、-C(O)NR⁷R⁸、-NHC(O)NHR⁶、-NHC(O)OR⁶、或-NHC(O)NHOR⁶的取代基所取代；R²選自烷基，其中該烷基進一步被一個或多個選自芳基或雜芳基的取代基所取代，其中該芳基或雜芳基視需要進一步被一個或多個選自鹵素、烷基、鹵烷基、氰基、硝基、-C(O)OR⁶、-C(O)NR⁷R⁸、-OC(O)NR⁷R⁸、-OR⁶、-NHS(O)_mR⁶、-NHC(O)R⁶或-NR⁷R⁸的取代基所取代，其中該鹵烷基較佳為三氟甲基；R³選自芳基或雜芳基，其中該芳基或雜芳基視需要進一步被一個或多個選自鹵素、烷基、鹵烷基、-OR⁶、-C(O)OR⁶、-OC(O)R⁶、-C(O)R⁶、-NR⁷R⁸、-OC(O)NR⁷R⁸、 -C(O)NR⁷R⁸、-NHS(O)_mR⁶、-NHC(O)R⁶、-NHC(O)OR⁶、-NHC(O)NHR⁶或-NHC(O)NHOR⁶的取代基所取代；R⁴選自烷基；R⁵選自氫原子、環烷基、雜環基、芳基、雜芳基、-OR⁵、-NR⁷R⁸或-NR⁷S(O)_mR⁶，其中該烷基、環烷基、雜環基、芳基或雜芳基視需要進一步被一個或多個選自鹵素、側氧基、烷基、鹵烷基、羥烷基、-OR⁶、-C(O)OR⁶、-OC(O)R⁶、-NR⁷S(O)_mR⁶、-S(O)_mR⁶、-C(O)R⁶或-NHC(O)R⁶的取代基所取代；R⁶選自氫原子、烷基、環烷基、雜環基、芳基或雜芳基，其中該烷基、環烷基、雜環基、芳基或雜芳基各自獨立地視需要進一步被一個或多個選自烷基、鹵素、羥基、烷氧基、環烷基、雜環基、芳基、雜芳基、羧酸或羧酸酯的取代基所取代；R⁷或R⁸各自獨立選自氫原子、烷基、環烷基、雜環基、芳基或雜芳基，其中該烷基、環烷基、雜環基、芳基或雜芳基各自獨立地視需要進一步被一個或多個選自烷基、鹵素、羥基、烷氧基、環烷基、雜環基、芳基、雜芳基、羧酸或羧酯的取代基所取代；或者，R⁷或R⁸與相連接的氮原子一起形成雜環基，其中該雜環基內含有一個或多個N、O或S(O)_m雜原子，並且該雜環基視需要進一步被一個或多個選自烷基、鹵素、羥基、烷氧基、環烷基、雜環基、芳基、雜芳基、羧酸或羧酸酯的取代基所取代； m為0、1或2；n為1、2、3或4；且p為0，1或2。 An object of the present invention is to provide a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, and a mixture thereof, and Its pharmaceutically acceptable salts:

Wherein: when G is N, D is C, and E is -CH-; when G is C, D and E are N; R ^{1 is} selected from alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl Or -OR ⁶ , wherein the alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl are each independently further optionally selected from one or more of halogen, cyano, nitro, alkyl, and halogen Alkyl, hydroxyalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ ,- NHC (O) NHR ⁶ , -NHC (O) OR ⁶ , or -NHC (O) NHOR ⁶ is substituted by a substituent; R ^{2 is} selected from an alkyl group, wherein the alkyl group is further selected from one or more aryl groups Or heteroaryl, wherein the aryl or heteroaryl is optionally further substituted with one or more members selected from halogen, alkyl, haloalkyl, cyano, nitro, -C (O) OR ^{6 , -C (O) NR 7 R} 8, -OC (O) NR 7 R 8, -OR 6, -NHS (O) m R 6, -NHC (O) R 6 or -NR ⁷ R ⁸ substituents of Substituted, wherein the haloalkyl group is preferably trifluoromethyl; R ^{3 is} selected from aryl or heteroaryl, wherein The aryl or heteroaryl group is further optionally selected from one or more of halogen, alkyl, haloalkyl, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -C (O ) R ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHS (O) _m R ⁶ , -NHC (O) R ⁶ , -NHC ( O) OR ⁶ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ are substituted by a substituent; R ^{4 is} selected from an alkyl group; R ^{5 is} selected from a hydrogen atom, a cycloalkyl group, a heterocyclic group, and an aryl group , Heteroaryl, -OR ⁵ , -NR ⁷ R ⁸ or -NR ⁷ S (O) _m R ⁶ , wherein the alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is further Or more selected from halogen, pendant oxygen, alkyl, haloalkyl, hydroxyalkyl, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NR ⁷ S (O) _m R ⁶ , -S (O) _m R ⁶ , -C (O) R ⁶ or -NHC (O) R ⁶ is substituted by a substituent; R ^{6 is} selected from a hydrogen atom, an alkyl group, a cycloalkyl group, and a heterocyclic group , Aryl or heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl are each independently further optionally selected from one or more of alkyl, halogen, hydroxy, alkoxy , Cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid or carboxylic acid ester substituents R ⁷ or R ⁸ are each independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each Optionally further substituted with one or more substituents selected from alkyl, halogen, hydroxy, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, or carboxylate, as necessary; or R ⁷ or R ⁸ forms a heterocyclic group together with the connected nitrogen atom, wherein the heterocyclic group contains one or more N, O or S (O) _m heteroatoms, and the heterocyclic group is further Substituted by one or more substituents selected from alkyl, halogen, hydroxy, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid or carboxylic acid ester; m is 0, 1 or 2; n is 1, 2, 3, or 4; and p is 0, 1, or 2.

其中：n，R¹至R⁵的定義如通式(I)中所述。 In a preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer And a mixture thereof, and a pharmaceutically acceptable salt thereof, which is a compound represented by the general formula (II) or a tautomer, meso, racemate, enantiomer, diastereomer Isomers and their mixtures, and their pharmaceutically acceptable salts:

Wherein: n, R ¹ to R ⁵ are as defined in the general formula (I).

其中：n，R¹至R⁵的定義如通式(I)中所述。 In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And its pharmaceutically acceptable salts, which are compounds represented by the general formula (III) or tautomers, mesomers, racemates, enantiomers, diastereomers Enantiomers and their mixtures, and their pharmaceutically acceptable salts:

Wherein: n, R ¹ to R ⁵ are as defined in the general formula (I).

In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And its pharmaceutically acceptable salts, wherein R ^{1 is} selected from aryl or heteroaryl, preferably phenyl or pyridazinyl, wherein the aryl or heteroaryl is further A plurality of substituents selected from the group consisting of halogen, cyano, nitro, alkyl, haloalkyl, hydroxyalkyl, or -OR ⁶ are substituted.

In another preferred embodiment of the present invention, a compound of the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And mixtures thereof, and pharmaceutically acceptable salts thereof, wherein R ^{2 is} selected from benzyl, wherein the benzyl is further optionally selected from one or more of halogen, alkyl, haloalkyl, cyano, nitro or -OR ⁶ is substituted by a substituent.

In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And its pharmaceutically acceptable salts, in which R ^{3 is} selected from aryl, preferably phenyl, wherein the aryl is optionally further selected from one or more -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ is substituted with a substituent, preferably -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And mixtures thereof, and pharmaceutically acceptable salts thereof, wherein R ^{4 is} selected from methyl.

In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And a mixture thereof, and a pharmaceutically acceptable salt thereof, wherein R ^{5 is} selected from a hydrogen atom and n is 1 or 2.

其中：n、D、E、G和R¹、R²、R⁴和R⁵的定義如通式(I)中所述；R^a選自烷基或-OR⁶，其中該烷基視需要進一步被一個或多個選自烷基、鹵素、羥基、烷氧基、環烷基、雜環基、芳基、雜芳基、羧酸或羧酸酯的取代基所取代；且R⁶選自烷基，其中該烷基視需要進一步被一個或多個選自烷基、鹵素、羥基、烷氧基、環烷基、雜環基、芳基、雜芳基、羧酸或羧酸酯的取代基所取代。 In another preferred embodiment of the present invention, a compound represented by the general formula (I) or a tautomer, meso, racemate, enantiomer, diastereomer And its pharmaceutically acceptable salts, which are compounds represented by the general formula (IV) or tautomers, mesomers, racemates, enantiomers, diastereomers Enantiomers and their mixtures, and their pharmaceutically acceptable salts:

Wherein: n, D, E, G and R ^1, in ^2, R ⁴ R ⁵ and R are as defined in general formula (I) a; R is selected from group ^A or -OR ^6, wherein the alkyl group optionally Further substituted with one or more substituents selected from the group consisting of alkyl, halogen, hydroxyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, or carboxylic acid ester; and R ^{6 is} selected Self-alkyl, where the alkyl is further optionally selected from one or more of alkyl, halogen, hydroxyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxylic acid, or carboxylic acid ester Is substituted with.

Typical compounds of the invention include, but are not limited to:

Or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof.

The invention provides a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and A method of a pharmaceutically acceptable salt, the method comprising:

A compound of the general formula (IA) is reacted with an amine represented by NH (R ⁴ ) (CH ₂ ) nR ⁵ and further reduced or / and tritiated if necessary to obtain a compound of the general formula (I); wherein: X is selected from halogen, The definitions of n, D, E, G and R ¹ to R ⁵ are as described in the general formula (I).

Reducing reagents include, but are not limited to, hydrogen or iron powder; tritiated reagents include, but are not limited to, carboxylic acids, fluorenyl chloride, sulfonium chlorochloride, methyl halide formate, isocyanate or triphosgene, and methoxyamine.

The present invention also provides a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, and a mixture thereof, and A method for its pharmaceutically acceptable salt, the method comprising:

The compound of the general formula (IB) is reacted with R ² X in the presence of a basic reagent, and further reduced or / and tritiated if necessary to obtain a compound of the general formula (I).

Wherein: X is selected from halogen, and n, D, E, G, and R ¹ to R ⁵ are defined as described in the general formula (I).

Basic reagents include, but are not limited to, organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 2,6-dimethylpyridine, sodium methoxide, lithium hexamethyldisilazide, Sodium hexamethyldisilazide, n-butyllithium, potassium tert-butoxide or tetrabutylammonium bromide, the inorganic bases include, but are not limited to, sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, carbonic acid Potassium hydrogen, cesium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide, preferably potassium carbonate or sodium methoxide; reducing reagents include, but are not limited to, hydrogen or iron powder; tritiation reagents include, but are not limited to, carboxylic acid, tritium chloride , Sulfonium chloride, methyl halide formate, isocyanate or triphosgene and methoxyamine.

其中：X選自鹵素；n、D、E、G和R¹至R⁵的定義如通式(I)中所述。 A compound represented by the general formula (IA) or (IB) or a pharmaceutically acceptable salt thereof as an intermediate for preparing the compound of the general formula (I),

Wherein: X is selected from halogen; n, D, E, G, and R ¹ to R ⁵ are as defined in general formula (I).

其中：D、E、G和R¹至R³的定義如通式(I)中所述。 A method for preparing a compound represented by general formula (IA) or a pharmaceutically acceptable salt thereof, which method comprises: performing a halogenation reaction on a compound of general formula (Ib) to obtain a compound of general formula (IA);

Wherein: D, E, G, and R ¹ to R ³ are as defined in the general formula (I).

其中：n、D、E、G和R¹、R³至R⁵的定義如通式(I)中所述； R^b選自烷基。 A method for preparing a compound represented by the general formula (IB) or a pharmaceutically acceptable salt thereof, the method comprising: a ring-forming reaction of a compound of the general formula (Ig) to obtain a compound of the general formula (IB);

Wherein: n, D, E, G, and R ¹ , R ³ to R ⁵ are as defined in the general formula (I); R ^{b is} selected from alkyl.

Typical compounds represented by general formula (IA) include, but are not limited to:

Or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof.

其中：n、D、E、G和R¹、R²、R⁴和R⁵的定義如通式(I)中所述。 A compound represented by the general formula (IVA) or a pharmaceutically acceptable salt thereof as an intermediate for preparing the compound of the general formula (IV),

Wherein: n, D, E, G and R ¹ , R ² , R ⁴ and R ⁵ are as defined in the general formula (I).

Typical compounds of general formula (IVA) include, but are not limited to:

Or tautomers, mesomers, racemates, enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof.

A method for preparing a compound of general formula (IV), the method comprising:

The compound of the general formula (IVA) is further reacted with a halogenating agent to obtain a compound of the general formula (IV); wherein: n, D, E, G and R ¹ , R ² , R ⁴ and R ⁵ are defined as the general formula (I) As described above; tritiated reagents include, but are not limited to, carboxylic acids, fluorenyl chloride, sulfonylchloride, methyl halide formate, isocyanate or triphosgene, and methoxyamine.

The invention further relates to a medicinal composition comprising A therapeutically effective amount of a compound represented by the general formula (I) of the present invention or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer, and a mixture thereof, And pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and Use of a pharmaceutically acceptable salt, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for a GnRH receptor antagonist.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and Use of a pharmaceutically acceptable salt, or a pharmaceutical composition containing the same, in the manufacture of a medicament for the treatment of gonadotropin-related diseases.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and A pharmaceutically acceptable salt, or a pharmaceutical composition containing the same, in the preparation or treatment of sex hormone-dependent cancers (e.g., prostate cancer, uterine cancer, breast cancer, pituitary cancer, etc.), bone metastases of sex hormone-dependent cancers, prostate hypertrophy, uterus Fibroids, endometriosis, uterine fibroids, precocious puberty, amenorrhea, premenstrual syndrome, dysmenorrhea, multi-atrial ovary syndrome, polycystic ovary syndrome, acne, hair loss, Alzheimer's disease , Infertility, allergic bowel syndrome, hormonal-independent and benign or malignant tumors or flushing diseases sensitive to LH-RH; used in the preparation of reproductive regulators, contraceptives, ovulation inducers use; Or it is used in the preparation of a medicament for preventing recurrence after surgery for the prevention of hormone-dependent cancer, preferably prostate cancer, uterine cancer, breast cancer, endometriosis or uterine fibroids.

The present invention also relates to a method for inhibiting GnRH receptor activity, which comprises administering a therapeutically effective amount of a compound represented by general formula (I) or a tautomer, meso, racemate, Enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them.

The present invention further relates to a method for treating a gonadotropin-related disease, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound represented by the general formula (I) or a tautomer, meso, racemate, Enantiomers, diastereomers and mixtures thereof, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them.

The invention further relates to a method for preventing or treating sex hormone-dependent cancers (prostate cancer, uterine cancer, breast cancer, pituitary cancer, etc.), bone metastases of sex hormone-dependent cancers, prostate hypertrophy, uterine fibroids, and endometriosis. , Uterine fibroids, precocious puberty, amenorrhea, premenstrual syndrome, dysmenorrhea, multi-atrial ovary syndrome, polycystic ovary syndrome, acne, hair loss, Alzheimer's disease, infertility, allergic bowel syndrome Of benign or malignant tumors or flushing diseases that are independent of hormones and sensitive to LH-RH, preferably prostate cancer, uterine cancer, breast cancer, endometriosis or uterine fibroids; used to regulate reproduction , Methods of contraception or inducing ovulation; or methods for preventing recurrence after surgery for sex hormone-dependent cancer.

The present invention also relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer thereof as a drug that inhibits the activity of the GnRH receptor. And its mixtures, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them.

The present invention also relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer thereof as a medicine for treating a gonadotropin-related disease. Structures and mixtures thereof, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them.

The present invention further relates to a compound represented by the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and A pharmaceutically acceptable salt, or a pharmaceutical composition containing the same, as a preventive or modulator of a hormone-dependent disease, and specifically, as a sex hormone-dependent cancer (for example, prostate cancer, uterine cancer, breast cancer, pituitary cancer Etc.), prostate hypertrophy, uterine fibroids, endometriosis, uterine fibroids, precocious puberty, amenorrhea syndrome, premenstrual syndrome, multi-room ovarian syndrome, acne, hair loss, Alzheimer's disease Prophylactic or therapeutic agents such as; used as pregnancy regulators (contraceptives, etc.), infertility medications or menstrual regulators; used as preventive or therapeutic agents for allergic bowel cancer syndrome; used as sex hormone dependence Preventive agents for recurrence of sexual cancer after surgery; preferably, prostate cancer, uterine cancer, breast cancer, endometriosis or uterine fibroids.

The active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide pleasing looks And delicious medicinal preparations. Tablets contain the active ingredients and non-toxic pharmaceutically acceptable excipients suitable for the preparation of tablets for mixing. These excipients can be inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, corn Starch or alginic acid; binders such as starch, gelatin, polyvinylpyrrolidone or gum arabic and lubricants such as magnesium stearate, stearic acid or talc. These tablets can be uncoated or they can be coated by known techniques that mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained release over a longer period. For example, water-soluble taste-masking substances, such as hydroxypropyl methyl cellulose or hydroxypropyl cellulose, or prolonged substances, such as ethyl cellulose, cellulose acetate butyrate, can be used.

Hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil vehicle such as peanut oil, liquid paraffin, or olive oil Soft gelatin capsules are provided as an oral preparation.

Aqueous suspensions contain the active substance and excipients suitable for the preparation of the aqueous suspension for mixing. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, and gum arabic; dispersants or wetting agents may be naturally occurring Phospholipids such as lecithin, or condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain fatty alcohols Compounds, such as heptadecaethyleneoxy cetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyethylene oxide sorbitol monooleate , Or the condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol anhydride, such as polyethylene oxide sorbitan monooleate. The aqueous suspension may also contain one or more preservatives such as ethyl paraben or n-propyl paraben, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents such as sucrose, Sweetener or Aspartame.

Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or a mineral oil such as liquid paraffin. The oil suspension may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. The sweeteners and flavoring agents described above can be added to provide a palatable formulation. These compositions can be preserved by the addition of antioxidants such as fenoxyfen or alpha-tocopherol.

By adding water, dispersible powders and granules suitable for use in the preparation of aqueous suspensions can provide active ingredients and dispersing or wetting agents, suspending agents or one or more preservatives for mixing. Suitable dispersing or wetting agents and suspending agents can illustrate the above examples. Other excipients such as sweeteners, flavors and colorants can also be added. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids such as soy lecithin and derived from fatty acids and hexitol anhydrides Esters or partial esters such as sorbitan monooleate, and condensation products of said partial esters and ethylene oxide, such as polyethylene oxide sorbitol monooleate. Emulsions may also contain sweeteners, flavoring agents, preservatives and antioxidants. Syrups and elixirs can be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol to form a microemulsion. Injections or microemulsions can be injected into a patient's bloodstream by local, large injections. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of a compound of the invention. To maintain this constant concentration, continuous intravenous drug delivery devices can be used. An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.

The pharmaceutical composition may be in the form of a sterile injectable water or oily suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol. In addition, a sterile fixed oil can be conveniently used as a solvent or suspension medium. For this purpose, any blended fixing oil including synthetic mono- or diesters can be used. In addition, fatty acids such as oleic acid can also be prepared for injection.

The invention can be administered in the form of suppositories for rectal administration Compound. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum and therefore will dissolve in the rectum to release the drug. Such materials include cocoa butter, glycerin gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the sex of the patient, the Diet, time of administration, mode of administration, rate of excretion, combination of drugs, etc. In addition, the optimal treatment such as the mode of treatment, the daily dosage of the general compound (I), or the type of pharmaceutically acceptable salt can be based on Traditional treatment options to verify.

Detailed description of the invention

Unless stated to the contrary, the following terms used in the specification and the scope of patent applications have the following meanings.

"Alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms. An alkyl group containing 1 to 10 carbon atoms is preferred, an alkyl group containing 1 to 6 carbon atoms is more preferred, an alkyl group containing 1 to 4 carbon atoms is most preferred, and a methyl group is most preferred. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, second butyl, n-pentyl, 1,1-dimethylpropyl Methyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl 2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl Methyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2- Dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethyl Hexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl- 3-ethylhexyl Group, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, third butyl, Dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Methyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Pentyl, 2,3-dimethylbutyl and the like. Alkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkyl and alkenyl , Alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, hetero Cycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendant oxygen, amine, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC ( O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

The term "alkenyl" refers to an alkyl group, as defined above, consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3 -Butenyl and the like. C _2-10 alkenyl is preferred, C _2-6 alkenyl is more preferred, and C _2-4 alkenyl is most preferred. Alkenyl may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkylthio , Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, pendant oxygen, amine, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C ( O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

The term "alkynyl" refers to an alkyl group, as defined above, consisting of at least two carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, 1-propynyl, 2-propynyl, 1-, 2- Or 3-butynyl and the like. C _2-10 alkynyl is preferred, C _2-6 alkynyl is more preferred, and C _2-4 alkynyl is most preferred. The alkynyl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkylthio , Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, pendant oxygen, amine, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C ( O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that includes 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably a cycloalkyl ring containing 3 to 10 carbon atoms, most preferably a cycloalkyl ring containing 3 to 6 carbon atoms, most preferably cyclopropyl. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptyl Alkenyl, cyclooctyl and the like are preferably cyclopropyl or cyclohexenyl. Polycyclic cycloalkyl includes spiro, fused and bridged cycloalkyl. Cycloalkyl can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, and alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio, pendant oxygen, amine, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ ,- NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that includes 3 to 20 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S (O) _m ( Where m is a heteroatom of the integer 0 to 2), excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. It preferably includes 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, more preferably the heterocyclyl ring contains 3 to 10 ring atoms, and even more preferably the heterocyclyl ring contains 5 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, tetrahydrofuranyl, and the like. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups. The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, and alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio, pendant oxygen, amine, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ ,- NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) group, preferably a 6 to 10 membered aryl group, having a conjugated pi-electron system , More preferably phenyl and naphthyl, and most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. Non-limiting examples include:

Aryl may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero Cycloalkylthio, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Heteroaryl" refers to a 5- to 14-membered all-carbon monocyclic or fused polycyclic group having a conjugated pi-electron system, further comprising 1 to 4 heteroatoms, wherein the heteroatoms are selected from one or more oxygen , Sulfur or nitrogen. 5 to 10 membered heteroaryl groups are preferred, 5 to 6 membered heteroaryl groups are more preferred, and furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, and pyrimidinyl are even more preferred , Pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. Non-limiting examples include:

Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, and alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkanethio Group, heterocycloalkylthio, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl and cycloalkyl are as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like. The alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkanethio Group, heterocycloalkylthio group, amino group, haloalkyl group, hydroxyalkyl group, carboxyl group, carboxylate group, -OR ⁶ , -C (O) OR ⁶ , -OC (O) R ⁶ , -NHS (O ) _m R ⁶ , -C (O) R ⁶ , -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NR ⁷ R ⁸ , -OC (O) NR ⁷ R ⁸ , -C (O) NR ⁷ R ⁸ , -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

"Haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above.

"Hydroxy" refers to the -OH group.

"Hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein the alkyl group is as defined above.

"Halogen" means fluorine, chlorine, bromine or iodine.

"Amino" means -NH _2.

"Cyano" refers to -CN.

"Nitro" refers to -NO _2.

"Side oxygen" means = O.

"Carboxylic acid" refers to (alkyl) or (cycloalkyl) -C (O) OH.

"Sulfonyl chloride" refers to (alkyl) or (cycloalkyl) -S (O) m-X (halogen).

"Isocyanate" means (alkyl) or (cycloalkyl) -N = C = O.

"Triphosgene" refers to bis (trichloromethyl) carbonate.

"Carboxy" refers to -C (O) OH.

"Carboxylate" refers to -C (O) O (alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

"As needed" or "as needed" means that the event or environment described later may, but need not, occur, and the description includes the place where the event or environment occurs or does not occur. For example, "heterocyclic group substituted with an alkyl group as necessary" means that the alkyl group may but need not exist, and the description includes a case where the heterocyclic group is substituted with an alkyl group and a case where the heterocyclic group is not substituted with an alkyl group .

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to 5 and more preferably 1 to 3 hydrogen atoms independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amine or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond.

"Pharmaceutical composition" means that it contains one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof and other A mixture of chemical components, as well as other components such as physiological / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the living body, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

Wherein R ⁶ to R ⁸ are as defined in the general formula (I).

Synthesis method of the present invention

In order to accomplish the synthetic purpose of the present invention, the present invention adopts the following synthetic technical scheme: a method for preparing a compound of the general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer Method for enantiomers and mixtures thereof, and pharmaceutically acceptable salts thereof, the method comprising:

The general formula (Ia) is reacted with R ² X in the presence of a basic reagent to obtain a compound of the general formula (Ib); the compound of the general formula (Ib) is subjected to a halogenation reaction under a catalyst condition to obtain a compound of the general formula (IA), and the catalyst is preferred Is azobisisobutyronitrile, and the halogenating reagent is preferably N-bromosuccinimide; a compound of the general formula (IA) is reacted with an amine represented by NH (R ⁴ ) (CH ₂ ) nR ^{5 to} obtain the general formula (I ) Compounds; wherein: X is selected from halogen, n, D, E, G, and R ¹ to R ⁵ are as defined in general formula (I) as described in general formula (I); R ^{3 is} preferably aromatic And more preferably a phenyl group, wherein the aryl group is further substituted with one or more substituents selected from -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ as necessary.

Preparation of a compound represented by general formula (I) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and a pharmaceutically acceptable form thereof A method of salt, the method comprising:

The general formula (Ic) is subjected to a halogenation reaction under a catalyst condition to obtain a compound of the general formula (Id). The catalyst is preferably azobisisobutyronitrile, and the halogenating reagent is preferably N-bromosuccinimide; the general formula (Id) The compound is reacted with an amine represented by NH (R ⁴ ) (CH ₂ ) nR ^{5 to} obtain a compound of general formula (Ie); the compound of general formula (Ie) is further heated under trifluoroacetic acid to obtain a compound of general formula (If) ; The compound of the general formula (If) is reacted with triphosgene and then the amine represented by R ¹ NH _{2 to} obtain the compound of the general formula (Ig); the compound of the general formula (Ig) is subjected to a ring-forming reaction under basic conditions to obtain the general formula ( IB) compounds; compounds of general formula (IB) react with R ² X in the presence of a basic reagent to obtain compounds of general formula (I); wherein: X is selected from halogen; Rb is selected from alkyl; n, D, E, G and R ¹ to R ⁵ are as defined in the general formula (I) as described in the general formula (I); and R ^{3 is} preferably an aryl group, more preferably a phenyl group, wherein the aryl group is further as required It is substituted with one or more substituents selected from -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ .

Preparation of a compound represented by general formula (II) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and a pharmaceutically acceptable form thereof A method of salt, the method comprising:

The compound of the general formula (IIa) is heated under the condition of trifluoroacetic acid to obtain a compound of the general formula (IIb); the compound of the general formula (IIb) is sequentially reacted with triphosgene and an amine represented by R ¹ NH _{2 to} obtain a compound of the general formula (IIc) ; The compound of the general formula (IIc) is subjected to a ring formation reaction under basic conditions to obtain a compound of the general formula (IId); the compound of the general formula (IId) is reacted with R ² X in the presence of a basic reagent to obtain the general formula (IIe) Compound; a compound of general formula (IIe) is subjected to a halogenation reaction under a catalyst condition to obtain a compound of general formula (IIA). The catalyst is preferably azobisisobutyronitrile, and the halogenating reagent is preferably N-bromosuccinimide; (IIA) A compound is reacted with an amine represented by NH (R ⁴ ) (CH ₂ ) nR ^{5 to} obtain a compound of general formula (II); wherein: X is selected from halogen; Rb is selected from alkyl; n, R ¹ to R ⁵ Is as defined in the general formula (I); R ^{3 is} preferably an aryl group, more preferably a phenyl group, wherein the aryl group is further selected from one or more if necessary by- NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ is substituted.

Preparation of a compound represented by general formula (III) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and a pharmaceutically acceptable form thereof A method of salt, the method comprising:

The general formula (IIIa) is reacted with triphosgene and then the amine represented by R ¹ NH _{2 to} obtain a compound of general formula (IIIb); the compound of general formula (IIIb) is reacted under basic conditions to obtain a compound of general formula (IIIc); The compound of formula (IIIc) is reacted with R ² X in the presence of a basic reagent to obtain a compound of general formula (IIId); the compound of general formula (IIId) is subjected to a halogenation reaction under a catalyst condition to obtain a compound of general formula (IIIA). Azobisisobutyronitrile is preferred, and the halogenating reagent is preferably N-bromosuccinimide; a compound of the general formula (IIIA) is reacted with an amine represented by NH (R ⁴ ) (CH ₂ ) nR ^{5 to} obtain the general formula ( III) compounds; wherein: X is selected from halogen; Rb is selected from alkyl; n, R ¹ to R ⁵ are as defined in general formula (I) as described in general formula (I); R ^{3 is} preferably An aryl group, more preferably a phenyl group, wherein the aryl group is further substituted with one or more substituents selected from -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ if necessary.

Preparation of a compound represented by general formula (IV) or a tautomer, a racemate, a racemate, an enantiomer, a diastereomer and a mixture thereof, and a pharmaceutically acceptable form thereof A method of salt, the method comprising:

The general formula (IVa) can be prepared by synthetic routes 1 to 4. The general formula (IVa) is reacted with R ² X in the presence of a basic reagent to obtain a compound of the general formula (IVb); the compound of the general formula (IVb) is in a catalyst. A halogenation reaction is performed under conditions to obtain a compound of the general formula (IVc). The catalyst is preferably azobisisobutyronitrile, and the halogenating reagent is preferably N-bromosuccinimide; the compound of the general formula (IVc) and NH (R ⁴ ) (CH ₂ ) nR ⁵ reacts to give a compound of the general formula (IVd); (IVd) compound in the presence of ammonium chloride, iron reduces the nitro group to an amine group to obtain a compound of the general formula (IVA); (IVA) The compound is further reacted with a halogenating agent to obtain a compound of general formula (IV); wherein: X is selected from halogen, and n, D, E, G, R ^a , R ¹ to R ⁵ are defined as general formula (I) Is defined in general formula (I).

In the above technical solution, the reagents that provide basic conditions include organic bases and inorganic bases. The organic bases include, but are not limited to, triethylamine, pyridine, 2,6-dimethylpyridine, sodium methoxide, and hexamethyldisilazane. Lithium amino, sodium hexamethyldisilazide, n-butyl lithium, potassium third butoxide or tetrabutyl ammonium bromide, the inorganic bases include but are not limited to sodium hydride, sodium carbonate, sodium bicarbonate , Potassium carbonate, potassium bicarbonate, cesium carbonate, lithium hydroxide, sodium hydroxide or potassium hydroxide; in the method of the present invention, the reagents with preferable basic conditions for the ring formation are organic bases, more preferably sodium methoxide.

Reducing reagents include, but are not limited to, hydrogen or iron powder; tritiated reagents include, but are not limited to, carboxylic acids, fluorenyl chloride, sulfonium chlorochloride, methyl halide formate, isocyanate or triphosgene, and methoxyamine.

The present invention is further described with reference to the following examples, but these examples do not limit the scope of the present invention.

The experimental methods without specific conditions in the examples of the present invention generally follow conventional conditions or conditions recommended by raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased on the market.

Examples

The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR chemical shifts (δ) are given in units of 10 ^-6 (ppm). The NMR measurement was performed using Bruker AVANCE-400 nuclear magnetometer. The measurement solvents were deuterated dimethylsulfinium (DMSO- d ₆ ), deuterated chloroform (CDCl ₃ ), and deuterated methanol (CD ₃ OD). The internal standard was four. Methylsilane (TMS).

MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

The HPLC was measured using an Agilent 1200DAD high-pressure liquid chromatography (Sunfire C18 150 × 4.6mm column) and a Waters 2695-2996 high-pressure liquid chromatography (Gimini C18 150 × 4.6mm column).

The average inhibition rate of the kinase and the IC ₅₀ value were measured using a NovoStar microplate reader (BMG, Germany).

The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silicone plate. The thin-layer chromatography (TLC) silicon plate uses a size of 0.15mm to 0.2mm. The thin-layer chromatography separation and purification product uses a size of 0.4mm to 0.5. mm.

Column chromatography generally uses Yantai Huanghai Silicone 200 ~ 300 mesh silica gel as the carrier.

The known starting materials of the present invention can be synthesized by or in accordance with methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Chemicals and other companies.

There are no special instructions in the examples, and the reactions can be performed under an argon atmosphere or a nitrogen atmosphere.

Argon or nitrogen atmosphere means that the reaction flask is connected to an approximately 1L Volume of argon or nitrogen balloon.

There is no special description in the examples, and the solution means an aqueous solution.

There is no special description in the examples, and the reaction temperature is room temperature, which is 20 ° C to 30 ° C.

The monitoring of the reaction progress in the examples uses thin layer chromatography (TLC). The developing systems used in the reaction are: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether And ethyl acetate system, D: acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound.

The eluent system for column chromatography and the eluent system for thin-layer chromatography for purifying compounds include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: dichloromethane and acetone System, D: ethyl acetate and dichloromethane system, E: ethyl acetate and dichloromethane and n-hexane, F: ethyl acetate and dichloromethane and acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound You can also add a small amount of basic or acidic reagents such as triethylamine and acetic acid to adjust.

Example 1

N- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) acetamidamine

first step 3-amino-5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester

3- (Third-butylamino) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1a (12.10 g, 34.93 mmol, Prepared by the method " Synthesis , 1988, (3), 203-207"), 100 mL of trifluoroacetic acid was added to a 250 mL eggplant-shaped flask, and the reaction was stirred at 85 ° C for 2.5 hours to stop the reaction. Cool to room temperature, distill under reduced pressure, add saturated sodium bicarbonate solution dropwise to adjust pH> 7, filter, dissolve the filter cake with dichloromethane, dry the organic layer with anhydrous sodium sulfate, filter, distill the filtrate under reduced pressure, and add 50 mL of the residue. A mixed solution of dichloromethane and methanol (V / V = 20: 1) was slurried, filtered, and dried to obtain 5.30 g of a yellow solid; the mother liquor was distilled under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to obtain the title. The product 3-amino-5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1b (combined to obtain 6.07 g, a yellow solid) in a yield of 60.1%.

MS m / z (ESI): 291.2 [M + 1]

Second step 3- (3- (2-fluoro-3-methoxyphenyl) ureido) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester

Dissolve 3-amino-5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1b (550 mg, 1.89 mmol) in 50 mL of dichloromethane, and Stirring does not completely dissolve. Add triethylamine (0.66 mL, 4.75 mmol) and triphosgene (225 mg, 0.76 mmol) and stir to dissolve. After stirring for 30 minutes, 2-fluoro-3-methoxyaniline (283 mg, 2 mmol) was added, and the mixture was stirred at room temperature for 12 hours to stop the reaction. Distilled under reduced pressure, the residue was added with 20 mL of water, and extracted with dichloromethane (50 mL × 2). The organic phases were combined, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure. A The resulting residue was purified to give the title product 3- (3- (2-fluoro-3-methoxyphenyl) ureido) -5-methyl-1- (4-nitrophenyl) -1 H- Pyrazole-4-carboxylic acid ethyl ester 1c (640 mg, white solid), yield 73.9%.

MS m / z (ESI): 458.1 [M + 1]

third step 5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

3- (3- (2-fluoro-3-methoxyphenyl) ureido) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1c (640 mg, 1.40 mmol) was added to 30 mL of an ethanol solution, sodium methoxide (190 mg, 3.52 mmol) was added, and the reaction was performed at room temperature for 3 hours. The reaction was heated to 80 ° C for 1 hour to stop the reaction. Cool to room temperature, add 3.5 mL of 1 M hydrochloric acid solution, and filter. The filter cake was washed with water (20 mL × 1), ethanol (10 mL × 1), methyl tert-butyl ether (5 mL × 1), and dried to give the title product. 5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H, 7 H) - -dione 1d (536mg, as a pale yellow solid), 93.2% yield.

MS m / z (ESI): 412.2 [M + 1]

the fourth step 7- (2,6-difluorobenzyl) -5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazole and [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4 , 6 (5 H , 7 H ) -dione 1d (530 mg, 1.29 mmol), 2- (chloromethyl) -1,3-difluorobenzene (230 mg, 1.41 mmol), potassium iodide (235 mg, 1.41 mmol), and Potassium carbonate (196 mg, 1.42 mmol) was added to 15 mL of N , N -dimethylformamide. The reaction was stopped at room temperature for 24 hours. Distilled under reduced pressure. The residue was stirred with 30 mL of water and 20 mL of methyl tert-butyl ether, filtered, the filter cake was dissolved with 50 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to obtain 570 mg of a solid. Liquid, the organic phase was washed with water (20mL × 3), saturated sodium chloride solution (20mL × 1), the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure. Tributyl ether (V / V = 1: 1) was washed and beaten, filtered, and the filter cake was dried to obtain 110 mg of a solid, which gave the title product 7- (2,6-difluorobenzyl) -5- (2-fluoro- 3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -Diketone 1e (680 mg, tan solid), yield 98.3%.

MS m / z (ESI): 538.2 [M + 1]

the fifth step 3- (bromomethyl) -7- (2,6-difluorobenzyl) -5- (2-fluoro-3-methoxyphenyl) -2- (4-nitrophenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

Under an argon atmosphere, 7- (2,6-difluorobenzyl) -5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 1e (660 mg, 1.23 mmol), azobisisobutyronitrile (24 mg, 0.15 mmol), and N -bromosuccinimide (262 mg, 1.47 mmol) was added to 20 mL of chlorobenzene, and the reaction was stopped at 85 ° C for 12 hours. Cool to room temperature, wash with saturated sodium chloride solution (15mL × 1), saturated sodium thiosulfate solution (15mL × 1), and extract the aqueous phase with dichloromethane (40mL × 2). Combine the organic phases with anhydrous Dry over sodium sulfate, filter, and distill the filtrate under reduced pressure to give the crude title product 3- (bromomethyl) -7- (2,6-difluorobenzyl) -5- (2-fluoro-3-methoxyphenyl) ) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 1f (900 mg, pale yellow solid), Used directly in the next step.

MS m / z (ESI): 616.1 [M + 1]

Step Six 7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -2- (4-nitrobenzene yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

The crude 3- (bromomethyl) -7- (2,6-difluorobenzyl) -5- (2-fluoro-3-methoxyphenyl) -2- (4-nitrophenyl)- 2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 1f (900 mg, 1.46 mmol) was dissolved in 3 mL of dimethylamine in a tetrahydrofuran solution and reacted at room temperature for 12 Hours, stop the reaction. Distilled under reduced pressure, and the obtained residue was purified by silica gel column chromatography using eluent system D to obtain 627 mg of crude product, which was purified by thin layer chromatography using developing solvent system E to obtain the title product 7- (2,6-difluoro Benzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -2- (4-nitrophenyl) -2 H -pyrazole and [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1g (223mg, as a pale yellow solid), yield: 31.0%.

MS m / z (ESI): 581.3 [M + 1]

Step Seven 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxybenzene yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -2- (4-nitro phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1g (212mg, 0.36mmol) dissolved in 8mL of formic acid was added 0.25mL 4 M A solution of HCl in 1,4-dioxane was added with 10% palladium on carbon (40 mg) and replaced with hydrogen three times. The reaction was carried out under normal pressure hydrogen for 2 hours to stop the reaction. Filter, filter cake was washed with methanol (20mL × 2), the organic phase was distilled under reduced pressure, 50mL of dichloromethane and 15mL of saturated sodium bicarbonate solution were added, stirred for 10 minutes, and the saturated sodium bicarbonate solution was added dropwise to adjust the pH of the aqueous phase> 7 , The layers were separated, and the aqueous phase was extracted with dichloromethane (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing system A to give the title. Product 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (180mg, white solid), yield: 89.5%.

MS m / z (ESI): 551.3 [M + 1]

Step eight N- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) acetamidamine

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (33mg, 0.059mmol) was dissolved in 5mL of dichloromethane, was added triethylamine amine (13 μ L, 0.094mmol) and acetic anhydride (7 μ L, 0.072mmol), 12 hours at room temperature, the reaction is not complete, supplemented with 5 μ L of acetic anhydride, was raised to 35 ℃ 2 hours, the reaction was stopped. Add 2 mL of saturated sodium bicarbonate solution, stir for 10 minutes, separate the layers, extract the aqueous phase with dichloromethane (15 mL x 3), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Use a thin layer for the residue Chromatographic purification with developing system A afforded the title product N- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro -3-methoxyphenyl) -4,6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) benzene ) Acetylamine 1 (25 mg, white solid), yield: 71.4%.

MS m / z (ESI): 593.1 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 7.89 (d, 2H), 7.67 (d, 2H), 7.36 (s, 1H), 7.25-7.30 (m, 1H), 7.19 (t, 1H), 7.08 (t, 1H), 7.89-7.94 (m, 3H), 5.45 (s, 2H), 3.93 (s, 3H), 3.69-3.70 (m, 2H), 2.35 (s, 6H), 2.25 (s, 3H).

Example 2

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea

first step 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (30mg, 0.054mmol) was added 5mL of dichloromethane, was added triethylamine (0.045mL, 0.32mmol) and triphosgene (6.5mg, 0.022mmol), react at room temperature for 1 hour, add methoxyamine (6.8mg, 0.081mmol), react at room temperature for 12 hours, add triphosgene (18mg, 0.061) mmol), reacted at 35 ° C for 1 hour, and then added methoxyamine (100 mg, 1.20 mmol), and reacted at 35 ° C for 2 hours to stop the reaction. 10 mL of saturated sodium bicarbonate was added, and the layers were separated. The aqueous phase was extracted with dichloromethane (15 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to thin-layer chromatography to develop the solvent. System A was purified to give the title product 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy Phenyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3- Methoxyurea 2 (12 mg, pale yellow solid), yield: 35.3%.

MS m / z (ESI): 624.5 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ). 7.90 (d, 2H), 7.71 (s, 1H), 7.67 (d, 2H), 7.20-7.30 (m, 3H), 7.06-7.08 (m, 1H), 6.89 -6.94 (m, 3H), 5.45 (s, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 3.86 (s, 2H), 2.35 (s, 6H).

Example 3

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethylurea

first step 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethylurea

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (26mg, 0.047mmol) was added 3mL of tetrahydrofuran, was added ethyl isocyanate (55 μ L, 0.69mmol), the reaction 35 ℃ 12 hours, the reaction was stopped. Distilled under reduced pressure, 10 mL of water and 20 mL of dichloromethane were added, and the layers were separated. The aqueous phase was extracted with dichloromethane (15 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Layer chromatography was purified with developing system A to give the title product 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2- (Fluoro-3-methoxyphenyl) -4,6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) Phenyl) -3-ethylurea 3 (12 mg, white solid), yield: 41.4%.

MS m / z (ESI): 622.5 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ). 7.80 (d, 2H), 7.45 (d, 2H), 7.22-7.26 (m, 2H), 7.13-7.15 (m, 1H), 6.99-7.04 (m, 1H) , 6.84-6.89 (m, 3H), 6.45 (s, 1H), 5.40 (s, 2H), 3.89 (s, 3H), 3.65 (s, 2H), 3.33-3.35 (m, 2H), 2.30 (s , 6H), 1.20 (t, 3H).

Example 4

(4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4,6- Dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) carbamate

first step (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4,6- Dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) carbamate

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (35mg, 0.064mmol) was added 5mL of dichloromethane, was added triethylamine (0.8 mL, 5.8 mmol) and methyl chloroformate (0.5 mL, 4.14 mmol) were reacted at 35 ° C for 12 hours, and the reaction was stopped. Lower to room temperature, add 10 mL of saturated sodium bicarbonate solution, separate the layers, extract the aqueous phase with dichloromethane (15 mL × 3), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Layer chromatography was purified with developing system A to give the title product (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro- 3-methoxyphenyl) -4,6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl ) Methyl carbamate 4 (24 mg, white solid), yield: 61.5%.

MS m / z (ESI): 609.5 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ). 7.88 (d, 2H), 7.55 (d, 2H), 7.25-7.27 (m, 1H), 7.18-7.20 (m, 1H), 7.06-7.08 (m, 1H) , 6.88-6.94 (m, 3H), 6.78 (s, 1H), 5.45 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 3.65-3.73 (m, 2H), 2.35 (s , 6H).

Example 5

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methylurea

first step 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methylurea

Acetic acid (46 mg, 0.77 mmol) was added to 1 mL of toluene, and N , N -diisopropylethylamine (0.2 mL, 1.13 mmol) and diphenyl azide phosphate (0.16 mL, 0.73 mmol) were added. Hour, cool in ice bath, add 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 1h (30mg, 0.050mmol) and 2 mL of dichloromethane was reacted at room temperature for 12 hours to stop the reaction. 10 mL of saturated sodium bicarbonate solution was added and extracted with dichloromethane (25 mL × 1). The organic phase was washed with water (10 mL × 1) and saturated sodium chloride solution (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced. The solution was concentrated under reduced pressure, and the residue was purified by thin layer chromatography using developing system A to give the title product 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) ) -5- (2-fluoro-3-methoxyphenyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] Pyrimidin-2-yl) phenyl) -3-methylurea 5 (8 mg, white solid), yield 24.4%.

MS m / z (ESI): 607.9 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 7.79 (d, 2H), 7.50 (d, 2H), 7.27-7.30 (m, 1H), 7.19 (t, 1H), 7.06 (t, 1H), 6.880-6.93 ( m, 4H), 5.44 (s, 2H), 4.95 (s, 1H), 3.92 (s, 3H), 3.78 (s, 2H), 2.88 (d, 3H), 2.39 (s, 6H).

Example 6

1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethyl Diurea

first step 5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

5- (2-fluoro-3-methoxyphenyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4 , 6 (5 H , 7 H ) -diketone 1d (1.90 g, 4.62 mmol), 2- (bromomethyl) -1-fluoro-3- (trifluoromethyl) benzene (1.31 g, 5.10 mmol), Potassium iodide (844 mg, 5.08 mmol) and potassium carbonate (702 mg, 5.09 mmol) were added to 54 mL of N , N -dimethylformamide, and stirred at room temperature for 20 hours to stop the reaction. Distilled under reduced pressure, added water and diethyl ether to beat for 1 hour, filtered, and the filter cake was washed with diethyl ether (10 mL × 3) and dried to give the title product 5- (2-fluoro-3-methoxyphenyl) -7- (2 -Fluoro-6- (trifluoromethyl) benzyl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 ( 5 H, 7 H) - dione 6a (1.91g, white solid) in a yield of 70.7%.

MS m / z (ESI): 58.1 [M + 1]

Second step 3- (bromomethyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2- (4-nitro phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -3-methyl-2- (4-nitrophenyl ) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 6a (1.81 g, 3.08 mmol), azobisisobutyronitrile (61 mg, 0.37 mmol ) And N -bromosuccinimide (660 mg, 3.71 mmol) were dissolved in 53 mL of chlorobenzene and replaced with argon three times. The reaction was stopped at 85 ° C for 19 hours under argon. A saturated sodium thiosulfate solution (30 mL × 1) and a saturated sodium chloride solution (50 mL × 2) were added to wash, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude title product 3- (bromomethyl)- 5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2- (4-nitrophenyl) -2 H -py pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 6b (2.18g, pale yellow solid), was used directly in the next step.

MS m / z (ESI): 666.0 [M + 1]

third step 3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

The crude 3- (bromomethyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2- (4- nitrophenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 6b (2.18g, 3.27mmol) and dimethylamine (2 M in tetrahydrofuran Solution) (6.8 mL 13.6 mmol) was added to the reaction flask, and stirred at room temperature for 12 hours to stop the reaction. Distilled under reduced pressure, and the residue was sent to prepare to give the title product 3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6 -(Trifluoromethyl) benzyl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 6c (455 mg, white solid), yield: 22.1%.

MS m / z (ESI): 630.8 [M + 1]

the fourth step 2- (4-aminophenyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- ( trifluoromethyl) benzyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl)- 2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 6c (200 mg, 0.32 mmol) dissolved in 8 mL of formic acid Then, 0.25 mL of a 4 M hydrochloric acid 1,4-dioxane solution was added, 10% Pd / C (50 mg) was added, and hydrogen was substituted for three times. The reaction was stopped at room temperature for 2 hours. Filter, wash the cake with a small amount of methanol, and concentrate the filtrate under reduced pressure. Add 50 mL of dichloromethane and 20 mL of sodium bicarbonate solution, stir for 10 minutes, adjust the pH of the aqueous phase to 8, and separate the liquid phase. Dichloromethane (25 mL × 2 ), Organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using developing system A to give the title product 2- (4-aminophenyl) -3- ((Dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2 H -pyridine pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - -dione 6d (135mg, white solid), yield: 71.1%.

MS m / z (ESI): 600.9 [M + 1]

the fifth step 1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazole [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethyl Urea

2- (4-Aminophenyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - -dione 6d (30mg, 0.050mmol) was dissolved in 1mL dichloro Ethyl isocyanate (0.06 mL, 0.76 mmol) was added to methane, and the reaction was stopped at 35 ° C for 12 hours. 5 mL of saturated sodium bicarbonate solution and 20 mL of dichloromethane were added, and the layers were separated. The organic phase was washed with water (10 mL × 1) and saturated sodium chloride solution (5 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing system A to give the title product 1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxy) Phenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazole [ 3,4- d ] pyrimidin-2-yl) phenyl) -3-ethylurea 6 (15 mg, white solid), yield: 44.1%.

MS m / z (ESI): 671.9 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 7.75 (d, 2H), 7.51 (d, 1H), 7.46 (d, 2H), 7.37-7.40 (m, 1H), 7.18-7.24 (m, 2H), 7.15 ( t, 1H), 6.91 (t, 1H), 6.85 (s, 1H), 5.60 (s, 2H), 4.92 (s, 1H), 3.90 (s, 3H), 3.69-3.76 (m, 2H), 3.28 -3.35 (m, 2H), 2.35 (s, 6H), 1.19 (t, 3H).

Example 7

1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Oxyurea

first step 1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Oxyurea

2- (4-Aminophenyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - -dione 6d (30mg, 0.050mmol) was dissolved in 3mL dichloro To methane, triethylamine (0.07 mL, 0.50 mmol) and triphosgene (15 mg, 0.051 mmol) were added and reacted for 1 hour at room temperature. Methoxyamine (25 mg, 0.30 mmol) was added to react for 1 hour at room temperature to stop the reaction. 10 mL of water was added, and extraction was performed with dichloromethane (25 mL × 1). The organic phase was sequentially washed with a saturated sodium bicarbonate solution (10 mL × 1), water (10 mL × 1), and a saturated sodium chloride solution (10 mL × 1). It was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing system A to give the title product 1- (4- (3-((dimethylamino) methyl)) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -4,6-dioxo-4,5,6 , 7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea 7 (12 mg, white solid), yield: 33.6%.

MS m / z (ESI): 673.8 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 7.85 (d, 2H), 7.64-7.70 (m, 3H), 7.53 (d, 1H), 7.39-7.42 (m, 1H), 7.23-7.28 (m, 3H), 7.06 (t, 1H), 6.92 (t, 1H), 5.57-5.65 (m, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 3.72 (s, 2H), 2.36 (s, 6H) .

Example 8

1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Diurea

first step 1- (4- (3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) Benzyl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Diurea

Acetic acid (62 mg, 1.03 mmol) was added to 1 mL of toluene, N , N -diisopropylethylamine (0.28 mL, 1.58 mmol) was added, and diphenyl azide phosphate (0.20 mL, 0.98 mmol) was added, and the reaction was performed at 70 ° C. 1.5 hours, 2- (4-aminophenyl) -3-((dimethylamino) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro -6- (trifluoromethyl) benzyl) -2 H -pyrazolo [3,4- d ] pyrimidin-4,6 (5 H , 7 H ) -dione 6d (30 mg, 0.050 mmol) and 1 mL Dichloromethane was reacted at room temperature for 12 hours, and the reaction was stopped. 5 mL of saturated sodium bicarbonate was added and extracted with dichloromethane (25 mL × 1). The organic phase was washed with water (10 mL × 1) and a saturated sodium chloride solution (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was decompressed. Concentrated, the residue was purified by thin-layer chromatography in developing system A to obtain the crude title product, and then purified by thin-layer chromatography in developing system A to give the title product 1- (4- (3-((dimethylamine (Methyl) methyl) -5- (2-fluoro-3-methoxyphenyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -4,6-dioxo- 4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methylurea 8 (13 mg, white solid), yield 39.4 %.

MS m / z (ESI): 658.2 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 鯋 7.76 (d, 2H), 7.51-7.53 (m, 1H), 7.47 (d, 2H), 7.39-7.40 (m, 1H), 7.24 (t, 1H), 7.19 (t, 1H), 7.04 (t, 1H), 6.89 (t, 1H), 6.74 (m, 1H), 5.56-5.64 (m, 2H), 4.85 (s, 1H), 3.92 (s, 3H), 3.77 (s, 2H), 2.88 (d, 3H), 2.38 (s, 6H).

Example 9

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4-d] pyrimidin-2-yl) phenyl) -3-methylurea

first step 5- (bromomethyl) -3- (third butylamino) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester

3- (Third-butylamino) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1a (23 g, 66.40 mmol), azobisiso Butyronitrile (2.18 g, 13.28 mmol), N -bromosuccinimide (14.18 g, 79.68 mmol) and 400 mL of chlorobenzene were added to a 1 L single-neck reaction flask and stirred well. Replace with argon three times, raise the temperature to 85 ° C, and react for 64 hours to stop the reaction. The reaction solution was concentrated under reduced pressure, 300 mL of dichloromethane was added, and the solution was washed with water (100 mL × 2), a saturated sodium thiosulfate solution (100 mL × 2), and a saturated sodium chloride solution (100 mL × 2), and dried over anhydrous sodium sulfate. , Filtered, and concentrated under reduced pressure to give the title product crude product 5- (bromomethyl) -3- (third butylamino) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid Ethyl 9a (25 g, brownish yellow oil) was used directly in the next reaction.

MS m / z (ESI): 427.1 [M + 2]

Second step 3- (Third-butylamino) -5-((dimethylamino) methyl) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester

Crude 5- (bromomethyl) -3- (third butylamino) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 9a (25 g, 58.78 mmol), A dimethylamine tetrahydrofuran solution (88 mL, 176.35 mmol) and 120 mL of tetrahydrofuran were added to a 500 mL single-necked reaction flask, and dissolved by stirring. After stirring at room temperature for 3 hours, the reaction was stopped. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using eluent system C to obtain the title product 3- (third butylamino) -5-((dimethylamino) methyl) -1 - (4-nitrophenyl) -1 H - pyrazole-4-carboxylate 9b (6g, brown oil), a yield of 26.2%.

MS m / z (ESI): 390.2 [M + 1]

third step 3-amino-5-((dimethylamino) methyl) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid methyl ester

3- (Third-butylamino) -5-((dimethylamino) methyl) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 9b (6g, 15.41 mmol) and 80 mL of trifluoroacetic acid were added to a 100 mL single-necked reaction flask and stirred well. The temperature was raised to reflux and the reaction was stopped for 1 hour. The reaction solution was concentrated under reduced pressure, 50 mL of dichloromethane was added, and a saturated sodium bicarbonate solution was added dropwise to adjust the pH to be greater than 7. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. To the residue were added 100 mL of methanol and 20 mL of dichloromethane, which were dissolved by stirring, potassium carbonate (6.37 g, 46.16 mmol) was added, and the mixture was stirred at room temperature for 17 hours. Filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using eluent system B to give the title product 3-amino-5-((dimethylamino) methyl) -1- (4-nitrate phenyl) -1 H - pyrazole-4-carboxylate 9c (3.30g, yellow solid), yield 67.1%.

MS m / z (ESI): 320.2 [M + 1]

the fourth step 5-((dimethylamino) methyl) -3- (3- (6-methoxypyridazin-3-yl) ureido) -1- (4-nitrophenyl) -1 H -py Azole-4-carboxylic acid methyl ester

Add 3-amino-5-((dimethylamino) methyl) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid methyl ester 9c (3.30 g, 10.33 mmol) and 100 mL of dichloromethane was added to a 250 mL single-necked reaction flask, and stirred well. Triethylamine (5.8 mL, 41.32 mmol) and triphosgene (1.84 g, 6.20 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 3-amino-6-methoxypyridazine (1.94g, 15.50mmol, prepared by the well-known method " Journal of Medicinal Chemistry , 2006,49 (14), 4409-4424"), and add the The reaction was stirred with warming for 2 hours, and the reaction was stopped. 200 mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL × 3). The organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluent system B to give the title product 5-((dimethylamine (Methyl) methyl) -3- (3- (6-methoxypyridazin-3-yl) ureido) -1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid methyl ester 9d (2.80 g, yellow solid), 57.6% yield.

MS m / z (ESI): 470.9 [M + 1]

the fifth step 3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4 -d ] pyrimidine-4,6 (5 H , 7 H ) -dione

5-((dimethylamino) methyl) -3- (3- (6-methoxypyridazin-3-yl) ureido) -1- (4-nitrophenyl) -1 H- Pyrazole-4-carboxylic acid methyl ester 9d (2.80 g, 5.95 mmol) and 120 mL of methanol were added to a 250 mL single-necked reaction flask, stirred well, and sodium methoxide (1.61 g, 29.76 mmol) was added. The temperature was raised to 50 ° C, and the reaction was stirred for 4 hours to stop the reaction. The reaction solution was concentrated under reduced pressure, 100 mL of water was added, and concentrated hydrochloric acid was added dropwise to adjust the pH to 5-6, and extracted with dichloromethane (100 mL × 3). The organic phase was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography to elute Agent System B was purified to give the title product 3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 9e (1.80g, yellow solid), yield 69.0%.

MS m / z (ESI): 438.9 [M + 1]

Step Six 7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrobenzene yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3, 4- d ] pyrimidine-4,6 (5 H , 7 H ) -diketone 9e (900mg, 2.05mmol) and 50mL N , N -dimethylformamide were added to a 100mL single-neck reaction flask, stirred well, added 2 , 6-difluorochlorobenzyl (367 mg, 2.26 mmol) and potassium carbonate (850 mg, 6.16 mmol). The temperature was raised to 40 ° C and the reaction was stirred for 17 hours to stop the reaction. 250 mL of water was added to the reaction solution, stirred for 20 minutes, filtered, and the filter cake was dried under vacuum to obtain the title product 7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- ( 6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H )- Dione 9f (1.10 g, yellow solid), yield 94.9%.

MS m / z (ESI): 564.9 [M + 1]

Step Seven 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazine-3- yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitro phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 9f (1g, 1.77mmol), 10 % palladium on carbon (200 mg of) and 80mL methanol Add to a 250mL single-necked reaction flask and stir well. Hydrogen was replaced 6 times and stirred at room temperature for 17 hours to stop the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the title product 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl)- 5- (6-methoxypyridazin-3-yl) -2 H -pyrazolo [3,4- d ] pyrimidin-4,6 (5 H , 7 H ) -dione 9 g (785 mg, yellow solid ), Yield 82.9%.

MS m / z (ESI): 534.9 [M + 1]

Step eight 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methylurea

Add acetic acid (483mg, 8.04mmol) and 15mL toluene to a 600mL autoclave, stir to dissolve, and add N , N -diisopropylethylamine (2.1mL, 12.07mmol) and diphenyl azide phosphate (2.22g, 8.07 mmol). The temperature was raised to 75 ° C, and the reaction was carried out for 1.5 hours. Add 10 mL of 2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxy yl pyridazin-3-yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 9g (480mg, 0.80mmol) in tetrahydrofuran was started and added The temperature was raised to 55 ° C., and the reaction was stopped for 16 hours. 20 mL of dichloromethane and 20 mL of water were added, and the layers were separated. The organic phase was sequentially washed with a saturated sodium bicarbonate solution (50 mL × 1), water (50 mL × 1), and a saturated sodium chloride solution (50 mL × 1), and then dried over anhydrous sodium sulfate. Dry, filter, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography using eluent system A to obtain a crude product, which was purified by thin layer chromatography using developing solvent system A to obtain a crude product (387 mg, brown solid). The crude yield was 81.3%. The two batches of crude product were combined, a total of 823 mg, purified by thin layer chromatography using developing system F. The resulting product was dissolved in methanol, concentrated under reduced pressure until a solid precipitated, and left to crystallize to give the title product 1- ( 4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4,6-di Pendantoxy-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methylurea 9 (450 mg, white solid) The total yield is 36.0%.

MS m / z (ESI): 592.3 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 8.17 (s, 1H), 7.54 (s, 1H), 7.42-7.45 (m, 4H), 7.24-7.30 (m, 2H), 6.88 (t, 2H), 5.95 ( s, 1H), 5.38 (s, 2H), 4.23 (s, 3H), 3.69 (s, 2H), 2.91 (s, 3H), 2.33 (s, 6H).

Example 10

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethylurea

first step 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-ethylurea

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazine-3 - yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 9g (22mg, 0.041mmol) was dissolved in 2mL of dichloromethane was added isocyanato Ethyl acetate (0.1 mL, 1.26 mmol) was reacted at 35 ° C for 12 hours, and the reaction was stopped. Add 5 mL of saturated sodium bicarbonate solution, extract with dichloromethane (30 mL × 1), wash the organic phase with water (10 mL × 1), saturated sodium chloride solution (10 mL × 1), dry over anhydrous sodium sulfate, filter, and filtrate It was concentrated under reduced pressure, and the residue was purified by thin layer chromatography in developing system A to give the title product 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) Yl) -5- (6-methoxypyridazin-3-yl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] Pyrimidin-2-yl) phenyl) -3-ethylurea 10 (10 mg, white solid), yield: 40.0%.

MS m / z (ESI): 606.2 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 8.07 (s, 1H), 7.55 (d, 1H), 7.38-7.43 (m, 4H), 7.25-7.28 (m, 2H), 6.858 (t, 2H), 5.87 ( t, 1H), 5.38 (s, 2H), 4.23 (s, 3H), 3.50 (s, 2H), 3.37 (t, 2H), 2.25 (s, 6H), 1.23 (t, 3H).

Example 11

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea

first step 1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4, 6-dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea

2- (4-aminophenyl) -7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazine-3 - yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 9g (100mg, 0.18mmol) was dissolved in 5mL of dichloromethane was added N, N -diisopropylethylamine (0.5 mL, 2.90 mmol) and triphosgene (46 mg, 0.15 mmol) were reacted at room temperature for 45 minutes. Methoxyamine hydrochloride (92 mg, 1.12 mmol) was added and reacted at 40 ° C for 12 hours. Stop reaction. 5 mL of saturated sodium bicarbonate solution was added and extracted with dichloromethane (25 mL × 1). The organic phase was washed with water (10 mL × 1) and saturated sodium chloride solution (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate. It was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developing agent system A to obtain a crude product in a combined batch of 62 mg of crude product. The thin layer chromatography was purified twice with developing agent system A to obtain the title product 1- (4- ( 7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4,6-dioxo -4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea 11 (45 mg, white solid), produced Rate: 24.7%.

MS m / z (ESI): 608.3 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 7.90 (d, 2H), 7.73 (s, 1H), 7.68 (d, 2H), 7.41 (d, 1H), 7.26-7.30 (m, 1H), 7.22 (s, 1H), 7.13 (s, 1H), 6.92 (t, 2H), 5.44 (s, 2H), 4.21 (s, 3H), 3.88 (s, 3H), 3.70 (s, 2H), 2.35 (s, 6H ).

Example 12

1- (4- (3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazine-3 -Yl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Diurea

first step 3- (3- (6-methoxypyridazin-3-yl) ureido) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester

Dissolve 3-amino-5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 1b (10.80 g, 37.20 mmol) in 500 mL of dichloromethane, and Add triethylamine (20 mL, 144 mmol) and triphosgene (5.20 g, 17.5 mmol) and react at room temperature for 1 hour. Add 3-amino-6-methoxypyridazine (6.60 g, 52.3 mmol) and react at room temperature. After 12 hours, the reaction was stopped. Before combining the samples, add 100 mL of water and separate the liquid. The organic phase was washed with water (100 mL × 1) and saturated sodium chloride (100 mL × 1) solution in that order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel Purified by column chromatography with eluent system C to give the title product 3- (3- (6-methoxypyridazin-3-yl) ureido) -5-methyl-1- (4-nitrobenzene yl) -1 H - pyrazole-4-carboxylic acid ethyl ester 12a (8.72g, pale yellow solid), yield: 53.0%.

Second step 5- (6-methoxypyridazin-3-yl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

3- (3- (6-methoxypyridazin-3-yl) ureido) -5-methyl-1- (4-nitrophenyl) -1 H -pyrazole-4-carboxylic acid ethyl ester 12a (8.72 g, 19.70 mmol) was dissolved in 150 mL of methanol, sodium methoxide (2.70 g, 50 mmol) was added, and the mixture was heated to 55 ° C for 12 hours to stop the reaction. Distilled under reduced pressure, adjusted to pH <7 by adding 1 M hydrochloric acid solution dropwise, filtered, and the filter cake was washed with water (30mL × 1), methanol (30mL × 1), and ether (20mL × 1) in that order, and dried under vacuum to give the crude title product 5 -(6-methoxypyridazin-3-yl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 ( 5 H, 7 H) - dione 12b (8.34mg, pale yellow solid), was used directly in the next step.

MS m / z (ESI): 396.0 [M + 1]

third step 7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

Dissolve 2- (bromomethyl) -1-fluoro-3- (trifluoromethyl) benzene (purchased from Sinopharm Group Chemical Reagent Co., Ltd., product number XW239870821) (308mg, 1.20mmol) in 20mL N , N -di methyl acyl amine, were added the crude 5- (6-methoxy-pyridazin-3-yl) -3-methyl-2- (4-nitrophenyl) -2 H - pyrazolo [3 , 4- d] pyrimidine -4,6 (5 H, 7 H) - dione 12b (395mg, 1mmol) and potassium carbonate (276mg, 2mmol), 2 hours at room temperature, the reaction was stopped. The reaction solution was poured into 100 mL of water, filtered, and the filter cake was dried to give the title product 7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -3-methyl-2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidin-4,6 (5 H , 7 H ) -dione 12c (465 mg, yellow (Solid), yield: 81.4%.

MS m / z (ESI): 570.0 [M-1]

the fourth step 3- (bromomethyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitro phenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione

7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -3-methyl-2- (4-nitrobenzene ) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 12c (465 mg, 0.81 mmol), azobisisobutyronitrile (13 mg, 0.081 mmol ), N -bromosuccinimide (174 mg, 0.97 mmol) and 20 mL of chlorobenzene were added to a 100 mL reaction flask, and the reaction was stopped at a constant temperature of 80 ° C. for 17 hours under an argon atmosphere. Distillation under reduced pressure gave the crude title product 3- (bromomethyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 12d (600 mg, yellow oil), Used directly in the next reaction.

MS m / z (ESI): 650.1 [M + 1]

the fifth step 3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

The crude 3- (bromomethyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2- (4- nitrophenyl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 12d (15mg, 0.033mmol) was dissolved in 20mL of tetrahydrofuran, was added 2 M A solution of dimethylamine (1 mL, 2.03 mmol) in tetrahydrofuran was reacted at room temperature for 2 hours to stop the reaction. Distilled under reduced pressure, and the residue was purified by thin-layer chromatography in developing system A to give the title product 3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl ) -5- (6-methoxypyridazin-3-yl) -2- (4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H, 7 H) - dione 12e (85mg, yellow solid), yield: 17.0%.

MS m / z (ESI): 615.2 [M + 1]

Step Six 2- (4-aminophenyl) -3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxy Pyridazin-3-yl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione

3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2 -(4-nitrophenyl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -dione 12e (100 mg, 0.14 mmol) dissolved in 5 mL of formic acid Then, 10% palladium on carbon (48 mg) was added, and hydrogen was replaced three times under normal pressure. The reaction was stopped at room temperature for 3 hours under a hydrogen atmosphere. The reaction solution was filtered, the filtrate was poured into 10 mL of water, the saturated sodium bicarbonate solution was added dropwise to adjust the pH> 7, and the mixture was extracted with dichloromethane (25 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing system A to give the title product 2- (4-aminophenyl) -3-((dimethylamino) methyl) -7- (2-fluoro-6- (Trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -2 H -pyrazolo [3,4- d ] pyrimidine-4,6 (5 H , 7 H ) -Diketone 12f (50 mg, white solid), yield: 53.0%.

MS m / z (ESI): 585.2 [M + 1]

Step Seven 1- (4- (3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazine-3 -Yl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methyl Diurea

Dissolve acetic acid (51mg, 085mmol) and N , N -diisopropylethylamine (0.22mL, 1.28mmol) in 2mL of toluene, add diphenyl azide phosphate (236mg, 0.86mmol), and react at 75 ° C for 1.5 hours 2 mL of 2- (4-aminophenyl) -3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6- methoxy-pyridazin-3-yl) -2 H - pyrazolo [3,4- d] pyrimidine -4,6 (5 H, 7 H) - dione 12f (15mg, 0.033mmol) in tetrahydrofuran, The reaction was stopped at 50 ° C for 12 hours. 10 mL of saturated sodium bicarbonate solution was added and extracted with dichloromethane (20 mL × 1). The organic phase was collected, washed with water (10 mL × 1) and saturated sodium chloride (10 mL × 1), and distilled under reduced pressure. Layer chromatography was purified with developing system A to give the title product 1- (4- (3-((dimethylamino) methyl) -7- (2-fluoro-6- (trifluoromethyl) benzyl) -5- (6-methoxypyridazin-3-yl) -4,6-dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] Pyrimidin-2-yl) phenyl) -3-methylurea 12 (20 mg, white solid), yield: 36.3%.

MS m / z (ESI): 642.3 [M + 1]

¹ H NMR (400MHz, CDCl ₃ ) 8.19 (s, 1H), 7.56 (d, 1H), 7.43-7.46 (m, 3H), 7.34-7.39 (m, 3H), 7.27 (d, 1H), 7.16 ( t, 1H), 6.05 (s, 1H), 5.48 (s, 2H), 4.22 (s, 3H), 3.48 (s, 2H), 2.93 (d, 3H), 2.21 (s, 6H).

Example 13

1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-ethylurea

first step ( E ) -4- (dimethylamino) -3- (4-nitrophenyl) -3-buten-2-one

Add 4-nitrophenylacetone 13a (3.8 g, 21.21 mmol) and N , N -dimethylformamide dimethyl acetal to a 50 mL flask, and react at 100 ° C for 30 minutes to stop the reaction. 150 mL of dichloromethane was added and stirred, and the obtained compound was purified by silica gel column chromatography using eluent system A to obtain the titled product ( E ) -4- (dimethylamino) -3- (4-nitrate Phenyl) 3-buten-2-one 13b (3 g, yellow solid), yield: 60.5%.

MS m / z (ESI): 235.0 [M + 1]

Second step 3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester

( E ) -4- (Dimethylamino) -3- (4-nitrophenyl) 3-buten-2-one 13b (1.50 g, 6.48 mmol) and 2-aminoacetamidine ethyl acetate in that order The ester hydrochloride (1.40 g, 7.78 mmol) was dissolved in 40 mL of acetic acid, and the reaction was carried out at room temperature for 16 hours, and the reaction was stopped at 100 ° C for 5 hours. Distilled under reduced pressure, added 120 mL of dichloromethane, washed with saturated sodium bicarbonate solution (50 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate under reduced pressure. The residue was slurried with 15 mL of ethyl acetate, filtered, and the filter cake was dried. to give the title product, 3-methyl-4- (4-nitrophenyl) -1 H - pyrrole-2-carboxylate 13c (412mg, yellow solid), yield 24.5%.

MS m / z (ESI): 273.1 [M-1]

third step 1-Amino-3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester

Dissolve 3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 13c (412 mg, 1.50 mmol) in 6 mL of N , N -dimethylformamide, ice Under bath conditions, sodium hydride (72 mg, 1.80 mmol) was added, and the mixture was reacted at room temperature for 30 minutes. 20 mL of a perchloramine solution was added, and the mixture was reacted at room temperature for 2 hours to stop the reaction. Add 50 mL of saturated sodium thiosulfate solution, stir for 10 minutes, separate the layers, extract the aqueous phase with ether (30 mL × 3), wash the organic phase with saturated sodium chloride solution (100 mL × 2), dry over anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure to give the title product 1-amino-3-methyl-4- (4-nitrophenyl) -1 H - pyrrole-2-carboxylate 13d (381mg, yellow solid), yield 87.7 %.

MS m / z (ESI): 290.2 [M + 1]

the fourth step 1- (3- (2-fluoro-3-methoxyphenyl) ureido) -3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester

Add 1d-amino-3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 13d (381mg, 1.30mmol) to 30mL of dichloromethane, stir to dissolve, add Triethylamine (328mg, 3.25mmol) and triphosgene (157mg, 0.53mmol), react at room temperature for 30 minutes, add 2-fluoro-3-methoxyaniline (202mg, 1.43mmol), react at room temperature for 2 hours, stop reaction. Filtration, the filter cake was washed with water (10 mL × 2) and ethyl acetate (2 mL × 3) in this order, and dried to give the title product 1- (3- (2-fluoro-3-methoxyphenyl) ureido) -3 - methyl-4- (4-nitrophenyl) -1 H - pyrrole-2-carboxylic acid ethyl ester 13e (296mg, yellow solid), yield 49.9%.

MS m / z (ESI): 457.3 [M + 1]

the fifth step 3- (2-fluoro-3-methoxyphenyl) -5-methyl-6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine- 2,4 (1 H , 3 H ) -dione

1- (3- (2-fluoro-3-methoxyphenyl) ureido) -3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 13e (290 mg, 0.64 mmol) and a solution of sodium hydroxide (128 mg, 3.20 mmol) in 10 ml of dichloromethane were reacted at 80 ° C for 2 hours to stop the reaction. After cooling to room temperature, 1 M hydrochloric acid solution was added dropwise to adjust the pH to 3 to 4, solids were precipitated, and the filter cake was washed with water (20 mL × 3) and ethanol (10 mL × 1) in this order to obtain the title product 3- (2- Fluoro-3-methoxyphenyl) -5-methyl-6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3H ) -diketone 13f (192 mg, grey solid), yield 73.3%.

MS m / z (ESI): 411.0 [M + 1]

Step Six 1- (2,6-difluorobenzyl) -3- (2-fluoro-3-methoxyphenyl) -5-methyl-6- (4-nitrophenyl) pyrrolo [2,1 -f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

3- (2-fluoro-3-methoxyphenyl) -5-methyl-6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine -2,4 (1 H , 3 H ) -dione 13f (192 mg, 0.47 mmol) and 2- (chloromethyl) -1,3-difluorobenzene (84 mg, 0.52 mmol) and potassium carbonate (162 mg, 1.75 mmol) was added to 10 mL of N , N -dimethylformamide. The reaction was stopped at 50 ° C for 16 hours. Distilled under reduced pressure. The residue was added with 20 mL of water and extracted with dichloromethane (20 mL x 1). The aqueous phase was extracted with dichloromethane (10 mL x 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure The residue was purified by thin layer chromatography using developing system A to give the title product 1- (2,6-difluorobenzyl) -3- (2-fluoro-3-methoxyphenyl) -5-methyl 6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13g (160mg, yellow solid ), Yield 63.7%.

MS m / z (ESI): 537.0 [M + 1]

Step Seven 5- (bromomethyl) -1- (2,6-difluorobenzyl) -3- (2-fluoro-3-methoxyphenyl) -6- (4-nitrophenyl) pyrrolo [ 2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

1- (2,6-difluorobenzyl) -3- (2-fluoro-3-methoxyphenyl) -5-methyl-6- (4-nitrophenyl) pyrrolo [2, 1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione 13 g (120 mg, 0.22 mmol), azobisisobutyronitrile (8 mg, 0.044 mmol), and N- Bromosuccinimide (47 mg, 0.26 mmol) was added to 5 mL of chlorobenzene, and the reaction was stopped at 85 ° C for 16 hours under an argon atmosphere. Cool to room temperature, distill under reduced pressure, add 10 mL of water to the residue, extract with dichloromethane (10 mL x 1), extract the aqueous phase with dichloromethane (10 mL x 3), collect the organic phase, and use a saturated sodium chloride solution ( 20mL × 1) was washed, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to give the title product 5- (bromomethyl) -1- (2,6-difluorobenzyl) -3- (2-fluoro- 3-methoxyphenyl) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -di Ketone 13h (130 mg, yellow solid), yield 96.2%.

MS m / z (ESI): 616.0 [M + 1]

Step eight 1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -6- (4-nitrobenzene ) Pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

The crude 5- (bromomethyl) -1- (2,6-difluorobenzyl) -3- (2-fluoro-3-methoxyphenyl) -6- (4-nitrophenyl) pyrrole And [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13h (130mg, 0.21mmol) and N , N -diisopropylethylamine ( 81 mg, 0.63 mmol) was dissolved in 5 mL of tetrahydrofuran, 1.6 mL of a solution of dimethylamine in tetrahydrofuran was added, and the reaction was stopped at 0-5 ° C for 1 hour. Distilled under reduced pressure, and the obtained residue was purified by thin layer chromatography using developing system A to give the title product 1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl)- 3- (2-fluoro-3-methoxyphenyl) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3H ) -diketone 13i (40 mg, yellow solid), yield: 33.1%.

MS m / z (ESI): 580.2 [M + 1]

Step Nine 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxybenzene ) Pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -6- (4-nitro Phenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione 13i (40 mg, 0.069 mmol), ammonium chloride (30 mg, 0.55 mmol) and iron (16 mg, 0.28 mmol) were dissolved in 5 mL of ethanol and water (V / V = 4: 1) mixed solvent, and reacted at 80 ° C. for 1 hour to stop the reaction. 20 mL of water was added and extracted with dichloromethane (10 mL × 3). The organic phase was collected, washed with a saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was used in a thin layer Chromatographic purification with developing system A provided the title product 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3 -(2-fluoro-3-methoxyphenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13j (20mg, Yellow solid), yield: 52.6%.

MS m / z (ESI): 550.2 [M + 1]

Step ten 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-ethylurea

6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxy Phenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13j (20mg, 0.036mmol) and ethyl isocyanate (13mg , 0.18 mmol) was added to a 30 mL sealed tube of 5 mL of tetrahydrofuran. The reaction was carried out at 50 ° C for 16 hours, the reaction was stopped, and the residue was distilled under reduced pressure. The residue was purified by thin-layer chromatography using developing system A to obtain the title product 1- (4- (1- (2,6-difluorobenzyl) -5 -((Dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2,4-dioxo-1,2,3,4-tetrahydropyrrolo [ 2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-ethylurea 13 (15 mg, white solid), yield: 68.1%.

MS m / z (ESI): 621.3 [M + 1]

¹ H NMR (400MHz, DMSO- d ₆ ). 7.87-7.77 (m, 4H), 7.69 (s, 1H), 7.58-7325 (m, 4H), 7.12-7.08 (m, 1H), 6.80-6.71 ( m, 1H), 4.42 (s, 2H), 3.83 (s, 3H), 3.45-3.50 (m, 2H), 3.20-3.31 (m, 2H), 2.16 (s, 6H), 1.12-1.03 (m, 3H)

Example 14

1- (4- (1- (2,6-difluorobenzyl-5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2,4 -Dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methylurea

first step 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methylurea

Add acetic acid (70 mg, 1.12 mmol) and N , N -diisopropylethylamine (0.32 mL, 1.80 mmol) to 1 mL of toluene, add diphenyl azide phosphate (0.25 mL, 1.20 mmol), and react at 70 ° C for 1.5 For 6 hours, add 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro 1-mL of -3-methoxyphenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13j (30mg, 0.054mmol) Dichloromethane solution was reacted at room temperature for 12 hours. 15mL of water was added and extracted with dichloromethane (30mL × 1). The organic phase was washed with saturated sodium bicarbonate (10mL × 1), water (10mL × 1), and saturated sodium chloride solution (10mL × 1) in that order, and dried with anhydrous After drying over sodium sulfate, filtering, and concentrating the filtrate under reduced pressure, the residue was purified twice by developing solvent using system A to give the title product 1- (4- (1- (2,6-difluorobenzyl) -5 -((Dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2,4-dioxo-1,2,3,4-tetrahydropyrrolo [ 2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methylurea 14 (9 mg, white solid), yield 27.3%.

MS m / z (ESI): 606.9 [M + 1]

¹ HNMR (400MHz, CDCl ₃ ) 8.86 (s, 1H), 7.55 (s, 2H), 7.24-7.35 (m, 3H), 7.68-7.14 (m, 6H), 6.21 (s, 1H), 5.60-5.74 (m, 2H), 4.54-4.59 (m, 2H), 3.97 (s, 3H), 2.77 (s, 3H), 2.55 (s, 6H).

Example 15

1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methoxyurea

first step 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxyphenyl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methoxyurea

6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (2-fluoro-3-methoxy Phenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 13j (55mg, 0.10mmol) was dissolved in 4mL of tetrahydrofuran, and Nitrophenyl chloroformate (30 mg, 0.15 mmol) and pyridine (32 mg, 0.40 mmol) were reacted at 30 ° C for 3 hours, and methoxyamine hydrochloride (25 mg, 0.30 mmol) was added. The reaction was sealed for 8 hours and stopped. reaction. Distilled under reduced pressure, and the residue was purified three times with developing system A by thin layer chromatography to give the title product 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino)) (Methyl) -3- (2-fluoro-3-methoxyphenyl) -2,4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1 , 2,4] triazin-6-yl) phenyl) -3-methoxyurea 15 (5 mg, yellow solid), yield: 8.1%.

MS m / z (ESI): 623.4 [M + 1]

¹ H NMR (400MHz, DMSO- d ₆ ) 7.90-7.87 (m, 2H), 7.85-7.80 (m, 2H), 7.60 (s, 1H), 7.55-7.25 (m, 4H), 7.09-7.02 (m , 1H), 6.71-6.65 (m, 1H), 4.50 (s, 2H), 3.84 (s, 3H), 3.67 (s, 3H), 3.54 (s, 2H), 2.15 (s, 6H)

Example 16

1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methylurea

first step 3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester

Dissolve 3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 13c (4.30 g, 15.70 mmol) in 200 mL of dichloromethane, and add triethylamine (21.7 in this order) mL, 157 mmol), 4-dimethylaminopyridine (192 mg, 1.57 mmol) and dicarbonyldi-tert-butyl ester (5.14 g, 23.50 mmol), and the reaction was stopped at room temperature for 12 hours. Distilled under reduced pressure, added 100 mL of water, extracted with dichloromethane (50 mL x 4), combined the organic phases, washed with saturated sodium chloride solution (150 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate under reduced pressure, leaving a residue The product was purified by silica gel column chromatography with eluent system B to give the title product 3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid third butyl-2- Ethyl formate 16b (4 g, yellow solid), yield: 68.1%.

MS m / z (ESI): 273.1 [M-100]

Second step 3- (bromomethyl) -4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester

3-methyl-4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester 16b (4g, 10.70mmol), azobisisobutyronitrile (351 mg, 2.14 mmol) and N -bromosuccinimide (2.28 g, 12.80 mmol) were dissolved in 40 mL of chlorobenzene, and the reaction was stopped at 80 ° C. for 16 hours under an argon atmosphere. Cooled to room temperature, distilled under reduced pressure, and dried under vacuum to give the title product 3- (bromomethyl) -4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl 2- Ethyl formate 16c (5.2 g, yellow solid) was used directly in the next step.

MS m / z (ESI): 453.0 [M-100]

third step 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester

3- (Bromomethyl) -4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester 16c (4.80 g, 10.70 mmol) was dissolved in 50 mL of tetrahydrofuran Then, a tetrahydrofuran solution of dimethylamine (26 mL, 53.50 mmol) was added, and the reaction was allowed to proceed at room temperature for 3 hours to stop the reaction. Distilled under reduced pressure and the residue was purified by silica gel column chromatography with eluent system A to give the title product 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H- Pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester 16d (3 g, yellow solid), yield: 67.4%.

MS m / z (ESI): 317.0 [M-100]

the fourth step 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester

3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-1-carboxylic acid tert-butyl-2-carboxylic acid ethyl ester 16d (3 g, 7.18 mmol) It was dissolved in 30 mL of a 2 M methanolic hydrochloric acid solution, and the reaction was stopped at room temperature for 3 hours. Distilled under reduced pressure, the residue was added with 30 mL of saturated sodium carbonate solution, and extracted with dichloromethane (30 mL × 4). The organic phase was collected, washed with saturated sodium chloride solution (60 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced. Pressure distillation gave the title product 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 16e (2 g, yellow solid) in a yield : 90.9%.

MS m / z (ESI): 318.1 [M + 1]

the fifth step 1-Amino-3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester

Dissolve 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 16e (1.8 g, 5.68 mmol) in 20 mL of N , N- To dimethylformamide, 60% sodium hydrogen (307 mg, 7.66 mmol) was added, and the mixture was reacted for 30 minutes at room temperature. 50 mL of chloramine was added, and the reaction was stopped at room temperature for 2 hours. 200 mL of water was added and extracted with ether (50 mL × 3). The organic phase was collected, washed with a saturated sodium chloride solution (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to give the title product 1-amino group. 3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 16f (620 mg, yellow solid), yield: 32.9%.

MS m / z (ESI): 333.2 [M + 1]

Step Six 3-((dimethylamino) methyl) -1- (3- (6-methoxypyridazin-3-yl) ureido) -4- (4-nitrophenyl) -1 H -pyrrole Ethyl-2-formate

1-Amino-3-((dimethylamino) methyl) -4- (4-nitrophenyl) -1 H -pyrrole-2-carboxylic acid ethyl ester 16f (420 mg, 1.26 mmol) was dissolved in 15 mL In dichloromethane, triethylamine (382 mg, 3.78 mmol), triphosgene (131 mg, 0.44 mmol), and 3-amino-6-methoxypyridazine (236 mg, 1.89 mmol) were added in this order, and the mixture was reacted at room temperature for 2 hours. , Stop the reaction. Distilled under reduced pressure, and the residue was purified by thin-layer chromatography in developing system A to give the title product 3-((dimethylamino) methyl) -1- (3- (6-methoxypyridazine-3- yl) ureido) -4- (4-nitrophenyl) -1 H - pyrrole-2-carboxylic acid ethyl ester 16g (298mg, yellow solid), yield: 30.2%.

Step Seven 5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [1 , 2,4] triazine-2,4 (1 H , 3 H ) -dione

3-((dimethylamino) methyl) -1- (3- (6-methoxypyridazin-3-yl) ureido) -4- (4-nitrophenyl) -1 H- 16 g (260 mg, 0.54 mmol) of pyrrole-2-carboxylic acid ethyl ester was dissolved in 10 mL of methanol, sodium methoxide (151 mg, 2.69 mmol) was added, and the reaction was stopped at 50 ° C for 3 hours. The distillation was performed under reduced pressure, and the residue was purified by thin layer chromatography with a developing system A to obtain the title product 5-((dimethylamino) methyl) -3- (6-methoxypyridazine-3- ) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione 16h (76mg, yellow (Solid), yield: 32.3%.

MS m / z (ESI): 438.3 [M + 1]

Step eight 1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -6- (4-nitrobenzene ) Pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [ 1,2,4] triazine-2,4 (1 H , 3 H ) -dione 16h (78mg, 0.18mmol), 2-chloromethyl-1,3-difluorophenyl (35mg, 0.21mmol) And potassium carbonate (37 mg, 0.26 mmol) were dissolved in 5 mL of N , N -dimethylformamide, and reacted at 50 ° C for 16 hours to stop the reaction. Distilled under reduced pressure, and the residue was purified by thin-layer chromatography in developing system A to give the title product 1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- ( 6-methoxypyridazin-3-yl) -6- (4-nitrophenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -Diketone 16i (72 mg, yellow solid), yield: 72.0%.

MS m / z (ESI): 564.1 [M + 1]

Step Nine 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazine-3- ) Pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione

1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -6- (4-nitro Phenyl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 (1 H , 3 H ) -dione 16i (72 mg, 0.13 mmol), iron (29 mg, 0.51 mmol) And ammonium chloride (55 mg, 1.02 mmol) were dissolved in 20 mL of ethanol and water (V / V = 4: 1) mixed solvent, reacted at 80 ° C for 1 hour, and the reaction was stopped. Cool to room temperature, add 20 mL of water, extract with dichloromethane (10 mL x 4), collect the organic phase, wash with saturated sodium chloride (10 mL x 1), dry over anhydrous sodium sulfate, filter, and distill the filtrate under reduced pressure. The resulting residue was purified by thin layer chromatography with developing system A to give the title product 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) (Methyl) -3- (6-methoxypyridazin-3-yl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -di Ketone 16j (25 mg, khaki solid), yield: 42.8%.

MS m / z (ESI): 534.3 [M + 1]

Step ten 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methylurea

Add acetic acid (51 mg, 0.84 mmol) to a 25 mL sealed tube, add 2 mL of toluene, N , N -diisopropylethylamine (163 mg, 1.26 mmol), and diphenyl azide phosphate (231 mg, 0.84 mmol) in order. The tube was reacted for 1 hour, cooled to 0 ° C, and 2 mL of 6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3 was added. -(6-methoxypyridazin-3-yl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 16j (45mg, 0.084 mmol) of methylene chloride solution, sealed at 40 ° C for 16 hours, and stopped the reaction. The distillation was performed under reduced pressure, and the residue was purified by thin-layer chromatography in developing system A to give the title product 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) ) -3- (6-methoxypyridazin-3-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1, 2,4] triazin-6-yl) phenyl) -3-methylurea 16 (18 mg, white solid), yield: 36.0%.

MS m / z (ESI): 591.3 [M + 1]

¹ H NMR (400MHz, DMSO- d ₆ ). 7.92-7.87 (m, 2H), 7.77-7.70 (m, 2H), 7.69 (s, 1H), 7.60-7.55 (m, 1H), 7.25-7.13 ( m, 2H), 7.05-6.99 (m, 1H), 6.79-6.71 (m, 1H), 4.48 (s, 2H), 4.06 (s, 3H), 3.54 (s, 2H), 2.75 (s, 3H) , 2.16 (s, 6H)

Example 17

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-hydroxypyridazin-3-yl) -4,6- Dioxo-4,5,6,7-tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea

first step

1- (4- (7- (2,6-difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-methoxypyridazin-3-yl) -4 , 6-Dioxo-4,5,6,7-tetrahydro-2 H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea 11 ( (122 mg, 0.20 mmol) was dissolved in 6 mL of tetrahydrofuran, stirred until completely dissolved, 40% hydrobromic acid (103 mg, 0.51 mmol) was added, and stirred at room temperature for 12 hours to stop the reaction. 5 mL of saturated sodium bicarbonate solution was added to the reaction solution, stirred for 5 minutes, extracted, and the organic phase was collected and purified by silica gel column chromatography with developing solvent A to obtain the title product 1- (4- (7- (2,6- Difluorobenzyl) -3-((dimethylamino) methyl) -5- (6-hydroxypyridazin-3-yl) -4,6-dioxo-4,5,6,7- Tetrahydro- 2H -pyrazolo [3,4- d ] pyrimidin-2-yl) phenyl) -3-methoxyurea 17 (16 mg, white solid), yield: 13.5%.

MS m / z (ESI): 594.3 [M + 1]

¹ H NMR (400MHz, DMSO- d ₆ ). 13.36-13.18 (m, 1H), 9.69 (s, 1H), 9.21-9.14 (m, 1H), 7.83-7.76 (m, 2H), 7.71-7.64 ( m, 2H), 7.52-7.46 (m, 1H), 7.46-7.36 (m, 1H), 7.09 (s, 3H), 5.32-5.21 (m, 2H), 3.71-3.62 (m, 5H), 2.17 ( s, 6H)

Example 18

1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazin-3-yl) -2, 4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1,2,4] triazin-6-yl) phenyl) -3-methoxyurea

first step

6- (4-aminophenyl) -1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) -3- (6-methoxypyridazine-3 -Yl) pyrrolo [2,1- f ] [1,2,4] triazine-2,4 ( 1H , 3H ) -dione 16j (170mg, 0.319mmol), 4-nitrophenylmethyl amino group-carboxylate (81mg, 0.382mmol, using patent application "WO 2011090935" obtained by the disclosed method of making), N, N - diisopropylethylamine (109 μ L, 0.638mmol) was dissolved in 10mL of tetrahydrofuran The reaction was stopped at room temperature for 4 hours. The distillation was performed under reduced pressure, and the residue was purified by thin-layer chromatography in developing system A to give the title product 1- (4- (1- (2,6-difluorobenzyl) -5-((dimethylamino) methyl) ) -3- (6-methoxypyridazin-3-yl) -2,4-dioxo-1,2,3,4-tetrahydropyrrolo [2,1- f ] [1, 2,4] triazin-6-yl) phenyl) -3-methoxyurea 18 (15 mg, yellow solid), yield: 7.8%.

MS m / z (ESI): 607.2 [M + 1]

Test example:

Biological evaluation

Test Example 1. Determination of the activity of the compound of the present invention on human-derived GnRHr (GnRH receptor)

GnRHr protein activity in vitro was tested by the following method.

This method is used to determine the inhibitory effect of the compounds of the present invention on the activity of human GnRHr protein expressed in human GnRHr / CHO stable transfected cells.

I. Experimental materials and instruments

1.Fluo-4 NW Calcium Assay Kits (F36206, invitrogen)

2.DMEM / F12 (SH30023.01B, thermo)

3.G418 (11811-031, invitrogen)

4.FlexStation3 microplate reader

Experimental steps

The mammalian expression vector containing human GnRHr gene was transferred into CHO cells with Lipofectamine ^® LTX reagent containing Plus ^TM ; antibiotic screening was started the next day to select monoclonal cell lines.

Human GnRHr / CHO stable transfected cells were seeded in 96-well plates at a density of 25,000 cells / well. The next day, the medium was removed, each well was added 100 μ L sample buffer containing the dye Fluo-4, 37 [deg.] C, incubated for 30 min. When the time is up, move the board to room temperature and equilibrate for 10 minutes. Each compound was formulated with 100 μ M DMSO, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M seven concentrations, each well was added 1 μ L, were incubated at room temperature 10 minutes. Performed with flexstation 3 microplate reader, automatically by the machine was added 50 μ L of GnRH polypeptide stimulation fluid, immediately reading at 494 / 516nM. The IC ₅₀ value of the compound can be calculated by software using fluorescence values corresponding to different concentrations.

The inhibitory activity of the compounds of the present invention on human-derived GnRHr was determined by the above test. The IC ₅₀ values measured are shown in Table 1.

Conclusion: The compound of the present invention has a significant inhibitory effect on human-derived GnRHr activity.

Test Example 2. The compound of the present invention is for monkey GnRHr Determination of inhibitory activity

This method is used to determine the inhibitory effect of the compound of the present invention on the monkey-derived GnRHr protein activity expressed in monkey-derived GnRHr / CHO stable transgenic cells.

I. Experimental materials and instruments

1.Fluo-4 NW Calcium Assay Kits (F36206, invitrogen)

2.DMEM / F12 (SH30023.01B, thermo)

3.G418 (11811-031, invitrogen)

4.FlexStation3 microplate reader

Experimental steps

The mammalian expression vector containing the monkey-derived GnRHr gene was transferred into CHO cells with Lipofectamine ^® LTX reagent containing Plus ^TM ; antibiotic screening was started the next day to select monoclonal cell lines.

Monkey-derived GnRHr / CHO stable transfected cells were seeded in 96-well plates at a density of 25,000 cells / well. The next day, the medium was removed, each well was added 100 μ L sample buffer containing the dye Fluo-4, 37 [deg.] C, incubated for 30 min. When the time is up, move the board to room temperature and equilibrate for 10 minutes. Each compound was formulated with 100 μ M DMSO, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M seven concentrations, each well was added 1 μ L, were incubated at room temperature 10 minutes. Flexstation 3 microplate reader with automatic addition of 50 μ L of solution GnRH polypeptide stimulation by a machine, immediately reading at 494 / 516nM. The IC ₅₀ value of the compound can be calculated by software using fluorescence values corresponding to different concentrations.

The inhibitory activity of the compounds of the present invention on monkey-derived GnRHr was determined by the above test. The measured IC ₅₀ values are shown in Table 2.

Conclusion: The compound of the present invention has obvious inhibitory effect on monkey-derived GnRHr activity.

Test Example 3. Determination of Rabbit Compound GnRHr Inhibitory Activity in the Invention

This method is used to determine the inhibitory effect of the compound of the present invention on the activity of rabbit-derived GnRHr protein expressed in rabbit-derived GnRHr / CHO stable transfected cells.

I. Experimental materials and instruments

1.Fluo-4 NW Calcium Assay Kits (F36206, invitrogen)

2.DMEM / F12 (SH30023.01B, thermo)

3.G418 (11811-031, invitrogen)

4.FlexStation3 microplate reader

Experimental steps

The mammalian expression vector containing the rabbit-derived GnRHr gene was transferred into CHO cells using Lipofectamine ^® LTX reagent containing Plus ^TM ; antibiotic screening was started the next day to select monoclonal cell lines.

Rabbit-derived GnRHr / CHO stable transfected cells were seeded in a 96-well plate at a density of 25,000 cells / well. The next day, the medium was removed, each well was added 100 μ L sample buffer containing the dye Fluo-4, 37 [deg.] C, incubated for 30 min. When the time is up, move the board to room temperature and equilibrate for 10 minutes. Each compound was formulated with 100 μ M DMSO, 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M seven concentrations, each well was added 1 μ L, were incubated at room temperature 10 minutes. Performed using Flexstation 3 microplate reader, automatically by the machine was added 50 μ L of GnRH polypeptide stimulation fluid, immediately reading at 494 / 516nM. The IC ₅₀ value of the compound can be calculated by software using fluorescence values corresponding to different concentrations.

The inhibitory activity of the compound of the present invention on rabbit-derived GnRHr was determined by the above test. The measured IC ₅₀ values are shown in Table 3.

Conclusion: The compound of the present invention has obvious inhibitory effect on rabbit-derived GnRHr activity.

Pharmacokinetic evaluation

Test Example 4, Rat Pharmacokinetic Tests of the Compounds of Example 2, Example 3, Example 5, Example 9, Example 10, Example 11 and Example 13 of the present invention

1.Abstract

SD rats were used as test animals, and rats were measured by LC / MS / MS method. After intragastric administration of the compound of Example 2, Example 3, Example 5, Example 9, Example 10, Example 11 and Example 13 and intravenous injection of the compound concentration of Example 11 at different times. The pharmacokinetic behavior of the compound of the present invention in rats was studied, and its pharmacokinetic characteristics were evaluated.

2. Test plan

2.1 Test drugs

Example 2, Example 3, Example 5, Example 9, Example 10, Example 11 and Example 13 Compounds

2.2 Test animals

Thirty-two healthy adult SD rats were divided into 8 groups of 4 males and 50 females. They were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2008-0016.

2.3 Drug Formulation

Weigh an appropriate amount of sample, add 0.5% CMC-Na, and make a 1mg / ml suspension by ultrasound; weigh an appropriate amount of drug, add 0.5ml DMSO and 0.5ml Tween 80 to dissolve, and then add physiological saline to the final volume.

2.4 Administration

Thirty-two SD rats were divided into eight groups of four males and half females. After fasting overnight, they were administered by gavage and intravenous injection at a dose of 10 mg / kg or 30 mg / kg by intravenous injection. The dose was 5 mg / kg, and the administration volume was 10 ml / kg.

3.Operation

0.1 ml of blood was collected before and 0.5, 1, 2, 4, 6, 8, 11, 24 hours after administration, placed in a heparinized test tube, centrifuged at 3500 rpm for 10 minutes, and the plasma was separated and stored at -20 ° C. Eat 2 hours after dosing.

LC / MS / MS method was used to determine the content of test compounds in rat plasma after intragastric administration.

4. Results of pharmacokinetic parameters

The pharmacokinetic parameters of the compounds of the invention are shown in Table 4 below: Conclusion: The compound of the present invention has good metabolism and absorption of drug by gastric administration in rats, and has obvious drug metabolism and absorption effect. In particular, Example 11 has good oral bioavailability.

Test example 5, rabbit pharmacokinetic test of Example 11 of the present invention

1.Abstract

Using rabbits as test animals, LC / MS / MS methods were used to determine the drug concentration in the plasma of rabbits given by gastric lavage and intravenous injection at different times after Example 11. The pharmacokinetic behavior of the compound of the present invention in rats was studied, and its pharmacokinetic characteristics were evaluated.

2. Test plan

2.1 Test drugs

Example 11 Compound

2.2 Test animals

New Zealand rabbits, 6 females, divided into 2 groups on average; purchased from Citrix.

2.3 Drug Formulation

Weigh an appropriate amount of sample, add 0.5% CMC-Na, and make a 3mg / ml suspension by ultrasonic; weigh an appropriate amount of drug, add 1ml DMSO and 1ml Tween 80 to dissolve, and then add physiological saline to the final volume.

2.4 Administration

New Zealand rabbits, 6 females, divided into 2 groups evenly. After fasting overnight, they were administered by gavage and intravenous injection. The dosage was 30 mg / kg, the volume was 5 ml / kg, and the intravenous injection was 5 mg. / kg, administration volume is 2ml / kg.

3.Operation

In the intravenous administration group, 0.2 ml of blood was collected at 0.25, 0.5, 1, 2, 4, 8, 11, 24 hours before and 5 minutes after the administration, and placed in a heparin anticoagulation test tube, and centrifuged at 3500 rpm for 10 minutes to separate the plasma , Stored at -20 ° C; blood was administered to the intragastric administration group before and 0.5,1,2,4,6,8,11,24 hours after administration. The treatment method was the same as that of the intravenous administration group. The LC / MS / MS method was used to determine the content of test compounds in rabbit plasma after intragastric and intravenous administration.

4. Results of pharmacokinetic parameters

The pharmacokinetic parameters of the compounds of the invention are shown in Table 5 below: Conclusion: The compound 11 of the present invention is well absorbed in rabbits and has good oral bioavailability.

Test example 6, canine pharmacokinetic test of Example 11 of the present invention

1.Abstract

The Beagle was used as the test animal, and the Beagle was measured by LC / MS / MS method. The concentration of drug in plasma at different times after canal administration and intravenous injection in dogs. The pharmacokinetic behavior of the compound of the present invention in Beagle dogs was studied, and its pharmacokinetic characteristics were evaluated.

2. Test plan

2.1 Test drugs

Example 11 Compound

2.2 Test animals

Eight healthy adult Beagle dogs were divided into two groups, male and female, purchased from Suzhou Xishan Zhongke Experimental Animal Co., Ltd.

2.3 Drug Formulation

Weigh an appropriate amount of the drug, add 0.5% CMC-Na, and make a 1.0 mg / ml suspension by ultrasonic; weigh an appropriate amount of the drug, add 5 ml of DMSO and 5 ml of Tween 80 to dissolve, and then add 90 ml of physiological saline to the final volume.

2.4 Administration

Eight healthy adult Beagle dogs were divided into two groups, male and female. After fasting overnight, they were administered by gavage and intravenous injection. The dosage of gavage was 5mg / kg. The volume of administration was 5ml / kg. The dose was 2 mg / kg, and the administration volume was 2 ml / kg.

3.Operation

In the intragastric administration group, 1.0 ml of blood was collected from venous veins before and after 0.25,0.5,1,2,4,6,8,12,24 hours, and placed in a heparin anticoagulation test tube, and centrifuged at 3500 rpm for 10 minutes. Plasma was separated and stored at -20 ° C; food was taken 2 hours after administration. In the intravenous administration group, before administration and 5 minutes after administration, 0.25, 0.5, 1, 2, Blood was collected at 4, 6, 8, 12, and 24 hours. The sample treatment process was the same as before. The content of test compounds in rabbit plasma was determined by LC / MS / MS after intragastric administration and intravenous administration.

4. Results of pharmacokinetic parameters

The pharmacokinetic parameters of the compounds of the invention are shown in Table 6 below: Conclusion: The compound 11 of the present invention is well absorbed in Beagle dogs and has good oral bioavailability.

Test Example 7 In vivo pharmacodynamic test of the compound of Example 11 of the present invention in rats

1.Abstract

A rabbit model of endometriosis was established, and a pharmacodynamic screening test for rabbit endometriosis using the compound of Example 11 was performed.

2. Test plan

2.1 Test drugs

Example 11 Compound

2.2 Test animals

15 New Zealand rabbits, female, divided into 3 groups evenly, purchased from Songlian Experimental Animal Farm, Songjiang District, Shanghai. Animal production license number: SYXK (Shanghai) 2013-0027.

2.3 Operation method

New Zealand rabbits are adaptively bred and quarantined for 7 days. Two consecutive days before surgery, subcutaneous injection of estradiol benzoate injection 30 μg / kg once a day; anesthetized with 3% sodium pentobarbital solution when the model is established, sterile laparotomy, free side Uterus, ligate the blood vessels in the mesentery, cut out a section of the uterus, cut the uterus longitudinally in a plate of (warm) physiological saline, separate the muscular layer and the endometrium, take a section of about 0.5cm × 0.5cm endometrium, and sew In the abdomen wall is rich in blood vessels, the intimal surface faces the abdominal cavity. Three weeks after grafting, laparotomy was performed to check the size of the endometrium of the transplantation. The 15 animals successfully modeled were randomly divided into 3 groups of 5 animals each, which were the vehicle control group and the low-dose compound of Example 11 (10mg / kg). ) Low and high dose groups (30mg / kg), with a dose volume of 5ml / kg, once / day, after intragastric administration for 28 days after successful modeling, body weight and ectopic endometrial volume were measured to evaluate the present invention Activity of the compound on the inner membrane.

2.4 Results:

A rabbit model of endometriosis was administered with the compound of Example 11 at a dose of 30 mg / kg for four weeks, and the same amount of 0.5% CMC-Na was administered to the stomach as a control, which could significantly inhibit the growth of ectopic endometrium at a dose of 10 mg / kg. The compound of Example 11 did not significantly inhibit the ectopic endometrial volume. Therefore, the rabbit of Example 11 The toxicological NOAEL dose (no harmful effect observed) of the endometriosis model is 30 mg / kg.

Test Example 8 Evaluation of 7-day toxicity and metabolic kinetics of the compound of Example 11 of the present invention in rats

1.Abstract

SD rats were used as test animals, and the compound of Example 11 was continuously administered by gavage for 7 days by LC / MS / MS. The toxicity of the compound to rodents was determined according to the possible toxic reaction and its severity. initial evaluation.

2. Test plan

2.1 Test drugs

Example 11 Compound

2.2 Test animals

Twenty-four healthy adult SD rats were divided into 4 groups of 6 males and half females, purchased from Shanghai Slark Experimental Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2012-0002.

2.3 Drug Formulation

Accurately weigh the compound of Example 11, add a small amount of 0.5% CMC-Na ultrasound or grind to a homogeneous suspension, add to the final volume and mix. Samples of the remaining solutions on Days 1 and 7 were retained for the concentration detection of the toxic metabolic drug solution.

2.4 Administration

Repeated gavage doses were 80, 240, and 720 mg / kg, and the concentrations were 8, 24, and 72 mg / ml, respectively. Dosing volume is 10ml / kg, once a day. 7 consecutive days.

3.Operation

Blood samples were collected on days 1 and 7 after administration. Blood collection time: blood samples were taken on day 1 before administration and 1, 2, 4, 8, and 24 hours after administration, and on day 7 before and after administration. Blood samples were collected at 1, 2, 4, 8, 24, and 48 hours after drug administration, plasma was separated by heparin anticoagulation, and stored at -20 ° C for testing. LC / MS / MS was used to determine the plasma levels of rabbit plasma after intragastric and intravenous administration. Content of the test compound.

4. Results

4.1. Impact on clinical signs 4.1.1 Death

The 720 mg / kg toxicity group of the compound of Example 11 died before administration on the 7th day; the 720 mg / kg toxicity group of the compound of Example 11 died before administration on the 7th day, and died on the 9th day on the 6th.

4.1.2 Clinical observation

During the administration, compared with the vehicle control group, 720 mg / kg of the compound of Example 11 on the 6th day of administration was toxic and the toxic metabolism group was wasted and the mental state was not good; 720 mg of the compound of Example 11 on the 7th day of administration Animals in the / kg toxicity group and the toxic metabolism group showed reduced food intake, weight loss, decreased activity, poor mental state, dull coat, shivering hair, arch back, blood around the nose and eyes, loose stools, and perianal contamination.

4.2. Impact on weight

Compared with the vehicle control group, on the 3rd and 7th days of administration, the body weight change of the compound of Example 11 in the high-dose group showed a significant decrease in%. (P <0.05).

4.3. Impact on food intake

Compared with the vehicle control group, the food intake of the 147280720mg / kg dose group was significantly reduced on the 3rd and 7th days.

4.4. Hematology test results

Compared with the vehicle control group, the animals of the compound of Example 11 in the 720 mg / kg dose group had WBC (white blood cells), Neut (relative and absolute values of neutrophils), Lymph (absolute value of lymphocytes), and EO (eosinophils). Absolute value) and RET (relative and absolute value of reticulocytes) decreased significantly (P <0.05).

4.5. Blood biochemical test results

Compared with the vehicle control group, the animals in the 720 mg / kg dose group of the compound of Example 11 had BUN (urea nitrogen), CHOL (total cholesterol), CREA (creatinine), TBIL (total bilirubin), TP (total protein), AST (Aspartate aminotransferase) was significantly increased (P <0.05), although there was no statistical difference in ALT (alanine aminotransferase), there were significant increases in two animals, ALB (albumin), ALP (Alkaline phosphatase) was significantly reduced (P <0.05). Due to the low blood volume of the animals in the SHR147280 720mg / kg dose group, the sample volume was insufficient, and the results of ion detection were missing.

4.6. Coagulation indicators

Compared with the vehicle control group, the compounds of Example 11 at the 80 mg / kg and 240 mg / kg dose groups had no significant differences. Because the animals in the SHR147280 720 mg / kg dose group had too little blood, the sample volume was insufficient, and the results of coagulation function indexes were missing.

4.7. Urine test

Compared with the vehicle control group, there was no regular abnormal change of urine index in each administration group.

4.8. Gross Pathological Results

Gross autopsy showed that compared with the vehicle control group, the compound of Example 11 in the 720mg / kg dose group had thymic bleeding and atrophy, spleen atrophy, and congestion, and various liver congestion and flaky gray-red necrosis in the liver leaves. , Pulmonary congestion, bleeding, adrenal bleeding, hypertrophy, ovarian bleeding and congestion, thinning of the gastrointestinal tract wall, white contents in the stomach, yellow contents of the small intestine, bleeding, colorectal bleeding, abdominal mucus-like; in addition, dead animals have necrotic foci. , The other groups were not seen with naked eyes.

4.9. Organ coefficient results

Compared with the vehicle control group, the absolute wet weight of the thymus, heart, lung and spleen of both sexes of the 720 mg / kg administration group of the compound of Example 11 was significantly reduced (p <0.05), and the absolute wet weight of the adrenal glands was significantly increased ( p <0.05). The relative organ coefficients of the thymus and spleen were significantly reduced (p <0.05), and the relative organ coefficients of the brain, liver, lung, kidney, and adrenal were significantly increased (p <0.05).

4.10. Results of toxic metabolic kinetic parameters

Note:-indicates that there is little data and the parameters cannot be calculated

Therefore, the toxicological NOAEL dose of Example 11 is 240 mg / kg, and the lethal dose is 720 mg / kg. The possible toxic target organs are the bone marrow hematopoietic system, thymus, heart, liver, lung, spleen, adrenal gland, ovary and digestive system.

To sum up: comparing the drug metabolism test of Test Example 5, the drug efficacy test of Test Example 7, and the toxic drug metabolism test of Test Example 8, the preliminary evaluation of the safety window of the compound of Example 11 is as follows:

Claims (25)

A compound represented by the general formula (I) or its tautomers, meso, racemates, enantiomers, diastereomers and mixtures thereof, and their pharmaceutically acceptable forms salt:

Where: when G is N, D is C, E is -CH-; when G is C, D and E are N; R ¹ is phenyl or pyridazinyl, the phenyl or pyridazinyl is independent of each other Optionally further substituted by one or more substituents selected from halogen, cyano, nitro, alkyl, haloalkyl, hydroxyalkyl or -OR ⁶ ; R ^{2 is} selected from C _1-6 alkyl, Wherein the C _1-6 alkyl group is further substituted with a phenyl group, wherein the phenyl group is further substituted with one or more selected from halogen, alkyl, haloalkyl, cyano, nitro or -OR ⁶ as needed Substituted by a group; R ³ is a phenyl group, the phenyl group is optionally further selected from one or more of -NHC (O) R ⁶ , -NHC (O) OR ⁶ , -NHC (O) NHR ⁶ or -NHC (O) substituted by a substituent of NHOR ⁶ ; R ^{4 is} selected from an alkyl group; R ^{5 is} selected from a hydrogen atom; R ^{6 is} selected from a hydrogen atom or an alkyl group; and n is 1, 2, 3, or 4. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and pharmaceutically acceptable salts thereof, wherein R ^{2 is} selected from C _1-6 alkyl, wherein the C _1-6 alkyl is further substituted by a phenyl group, wherein the phenyl group is further substituted by one or more Replaced by a trifluoromethyl group. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and its pharmaceutically acceptable salts, it is a compound represented by the general formula (II) or its tautomer, meso, racemate, enantiomer, diastereomer Body and its mixture, and its pharmaceutically acceptable salts:

Among them: n, R ¹ to R ⁵ are defined as described in item 1 of the patent application scope. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and its pharmaceutically acceptable salts, it is a compound represented by the general formula (III) or its tautomer, racemate, racemate, enantiomer, diastereomer Body and its mixture, and its pharmaceutically acceptable salts:

Among them: n, R ¹ to R ⁵ are defined as described in item 1 of the patent application scope. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and pharmaceutically acceptable salts thereof, wherein R ^{2 is} selected from benzyl, wherein the benzyl is optionally further selected from one or more selected from halogen, alkyl, haloalkyl, cyano, nitro or -OR ⁶ is substituted by a substituent. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and pharmaceutically acceptable salts thereof, wherein the R ³ is a phenyl group, wherein the phenyl group is optionally further substituted with one or more selected from -NHC (O) NHR ⁶ or -NHC (O) NHOR ⁶ Substituted. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and pharmaceutically acceptable salts thereof, wherein R ^{4 is} selected from methyl. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and a pharmaceutically acceptable salt thereof, wherein R ^{5 is} selected from a hydrogen atom and n is 1 or 2. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer and the In the form of a mixture, and its pharmaceutically acceptable salts, it is a compound represented by the general formula (IV) or its tautomer, racemate, racemate, enantiomer, diastereomer Body and its mixture, and its pharmaceutically acceptable salts:

Where: n, D, E, G, R ¹ , R ² , R ⁴ and R ⁵ are as defined in item 1 of the patent application scope; R ^{a is} selected from alkyl or -OR ⁶ ; and R ^{6 is} selected from alkyl. The compound represented by general formula (I) or its tautomer, meso, racemate, enantiomer, diastereomer as described in any one of the items 1 to 8 of the patent application scope Enantiomers and their mixtures, and their pharmaceutically acceptable salts, wherein the compound is selected from:

A method for preparing a compound represented by general formula (I) as described in item 1 of the patent application scope or its tautomer, meso, racemate, enantiomer and diastereomer And mixtures thereof, and methods of their pharmaceutically acceptable salts, which include:

The compound of general formula (IA) is reacted with the amine represented by NH (R ⁴ ) (CH ₂ ) nR ⁵ , and further reduction or / and acylation reaction is needed as needed to obtain the compound of general formula (I); The definitions of n, D, E, G and R ¹ to R ⁵ are as described in item 1 of the patent application scope. A method for preparing a compound represented by general formula (I) as described in item 1 of the patent application scope or its tautomer, meso, racemate, enantiomer and diastereomer And mixtures thereof, and methods of their pharmaceutically acceptable salts, which include:

The compound of general formula (IB) reacts with R ² X in the presence of an alkaline reagent, and if necessary, further reduction or / and acylation reaction, to obtain the compound of general formula (I); wherein: X is selected from halogen, n, D, E , G and R ¹ to R ⁵ are defined as described in item 1 of the patent application scope. A compound represented by general formula (IA) or a pharmaceutically acceptable salt thereof, which is used as an intermediate for preparing a compound represented by general formula (I),

Wherein: X is selected from halogen; D, E, G and R ¹ to R ³ are defined as described in item 1 of the patent application scope. A compound represented by general formula (IB) or a pharmaceutically acceptable salt thereof, which is used as an intermediate for preparing a compound represented by general formula (I),

Among them: n, D, E, G and R ¹ , R ³ to R ⁵ are defined as described in item 1 of the patent application scope. A method for preparing a compound represented by general formula (IA) or a pharmaceutically acceptable salt thereof as described in item 13 of the scope of patent application ) Compound;

Among them: D, E, G and R ¹ to R ³ are defined as described in item 1 of the patent application scope. A method for preparing a compound represented by general formula (IB) or a pharmaceutically acceptable salt thereof as described in item 14 of the scope of patent application, the method includes: a ring formation reaction of a compound of general formula (Ig) to obtain general formula (IB ) Compound;

Wherein: n, D, E, G and R ¹ , R ³ to R ⁵ are as defined in item 1 of the patent application scope; R ^{b is} selected from alkyl. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

A pharmaceutical composition containing a therapeutically effective amount of a compound represented by general formula (I) as described in any one of claims 1 to 10, or a tautomer or a racemate thereof , Racemates, enantiomers, diastereomers and their mixtures, and their pharmaceutically acceptable salts, as well as one or more pharmaceutically acceptable carriers, diluents or excipients. A compound using the compound represented by general formula (I) as described in any one of claims 1 to 10 or its tautomer, meso, racemate, enantiomer, The use of diastereomers and their mixtures, and their pharmaceutically acceptable salts, or the pharmaceutical composition as described in item 19 of the patent application scope in the preparation of a medicament as a GnRH receptor antagonist. A compound using the compound represented by general formula (I) as described in any one of claims 1 to 10 or its tautomer, meso, racemate, enantiomer, The use of diastereomers and their mixtures, and their pharmaceutically acceptable salts, or the pharmaceutical composition as described in item 19 of the patent application scope, in the preparation of a medicament for the treatment of diseases related to gonadal hormones. A compound using the compound represented by general formula (I) as described in any one of claims 1 to 10 or its tautomer, meso, racemate, enantiomer, Diastereomers and their mixtures, as well as their pharmaceutically acceptable salts, or the pharmaceutical composition as described in item 19 of the patent application, in the preparation of a treatment or prevention of sex hormone-dependent cancer, sex hormone-dependent cancer bone metastasis , Prostate hypertrophy, uterine fibroids, endometriosis, uterine fibroids, precocious puberty, amenorrhea, premenstrual syndrome, dysmenorrhea, multilocular ovarian syndrome, polycystic ovarian syndrome, acne, alopecia, Use of drugs for Alzheimer's disease, infertility, allergic bowel syndrome, hormone-independent and LH-RH sensitive benign or malignant tumors or flushing diseases. The use according to item 22 of the patent application scope, wherein the sex hormone-dependent cancer is selected from prostate cancer, uterine cancer, breast cancer, endometriosis or uterine fibroids. A compound of formula (I) or its tautomer, meso, racemate, enantiomer, or The use of enantiomers and their mixtures, as well as their pharmaceutically acceptable salts, or the pharmaceutical composition as described in item 19 of the patent application, in the preparation of drugs for reproduction regulators, contraceptives, and ovulation inducers. A compound using the compound represented by general formula (I) as described in any one of claims 1 to 10 or its tautomer, meso, racemate, enantiomer, The use of diastereomers and their mixture forms, and their pharmaceutically acceptable salts, or the pharmaceutical composition as described in item 19 of the patent application scope for the preparation of a medicament for preventing recurrence after surgery for sex hormone-dependent cancer, in which The sex hormone dependent cancer is selected from prostate cancer, uterine cancer, pituitary cancer, breast cancer, endometriosis or uterine fibroids.